U.S. patent application number 13/518876 was filed with the patent office on 2012-12-20 for lentivirus vaccine based on the recombinant viral vaccine against yellow fever.
Invention is credited to Myrna Cristina Bonaldo, Ricardo Galler, Jonah Bradley Sacha, David Ian Watkins.
Application Number | 20120321655 13/518876 |
Document ID | / |
Family ID | 44763608 |
Filed Date | 2012-12-20 |
United States Patent
Application |
20120321655 |
Kind Code |
A1 |
Bonaldo; Myrna Cristina ; et
al. |
December 20, 2012 |
LENTIVIRUS VACCINE BASED ON THE RECOMBINANT VIRAL VACCINE AGAINST
YELLOW FEVER
Abstract
The present invention relates to an attenuated, recombinant
viral vaccine against yellow fever which expresses heterologous
sequences of a lentivirus and is used as an immunisation agent to
induce an immune response to lentivirus.
Inventors: |
Bonaldo; Myrna Cristina;
(Jacarepagua, BR) ; Galler; Ricardo; (Pendotiba,
BR) ; Watkins; David Ian; (Arena, WI) ; Sacha;
Jonah Bradley; (Madison, WI) |
Family ID: |
44763608 |
Appl. No.: |
13/518876 |
Filed: |
December 22, 2010 |
PCT Filed: |
December 22, 2010 |
PCT NO: |
PCT/BR10/00430 |
371 Date: |
September 6, 2012 |
Current U.S.
Class: |
424/188.1 ;
424/199.1; 435/69.3; 530/350; 536/23.72 |
Current CPC
Class: |
A61K 2039/542 20130101;
A61K 2039/70 20130101; Y02A 50/388 20180101; C12N 2770/24143
20130101; A61K 2039/55594 20130101; A61P 31/18 20180101; C12N
2740/15034 20130101; A61K 2039/523 20130101; C12N 2770/24134
20130101; A61K 2039/545 20130101; A61K 39/12 20130101; A61K
2039/5256 20130101 |
Class at
Publication: |
424/188.1 ;
530/350; 536/23.72; 424/199.1; 435/69.3 |
International
Class: |
A61K 39/21 20060101
A61K039/21; C12P 21/02 20060101 C12P021/02; A61P 31/18 20060101
A61P031/18; C07K 14/155 20060101 C07K014/155; C12N 15/49 20060101
C12N015/49 |
Goverment Interests
[0001] Part of the described material has been developed with the
financial support of the National Institutes of Health of the
United States of America under number [identification code] R11
AI076114. Thus, the North American government may have certain
rights concerning the described technical material.
Foreign Application Data
Date |
Code |
Application Number |
Dec 23, 2009 |
BR |
PI0914507-9 |
Claims
1. A pharmaceutical composition for infection caused by
lentiviruses, comprising: (A) a recombinant yellow fever virus
which expresses a heterologous polypeptide comprising at least a
fragment of a lentiviral polypeptide, and (B) a pharmaceutical
carrier; wherein the composition comprises the recombinant yellow
fever virus in an amount effective to induce a therapeutic or
protective immune response against infection with lentivirus.
2. The composition according to claim 1, wherein the heterologous
polypeptide includes at least a fragment of an HIV Gag polypeptide
or at least a fragment of athe polypeptide of SIV Gag.
3. The composition according to claim 1, wherein the heterologous
polypeptide includes at least a fragment of SEQ ID NO:9.
4. The composition according to claim 1, wherein the fragment
comprises at least 8, 9, 10, 20, 30, 40, 50, 100 or 200 continuous
amino acids continuous of SEQ ID NO:9.
5. The composition according to claim 1, wherein the fragment
comprises amino acids 45-269 of SEQ ID NO:9.
6. The composition according to claim 1, wherein the heterologous
polypeptide comprises a sequence of amino acid C-terminal
comprising KESSIG.
7. The composition according to claim 1, wherein the recombinant
yellow fever virus is YF17D.
8. The composition according to claim 1, wherein the recombinant
yellow fever virus comprises a sequence coding for the heterologous
polypeptide inserted between sequences coding for polypeptide and
NS1 polypeptide.
9. The composition according to claim 1 characterized in that the
recombinant yellow fever virus comprises a sequence coding for the
heterologous polypeptide, said sequence being optimized according
to codon usage frequency of the yellow fever virus.
10. A pharmaceutical composition for infection caused by
lentiviruses, comprising a mixture of recombinant yellow fever
viruses, wherein the mixture comprises: (A) a first recombinant
yellow fever virus which expresses a first heterologous polypeptide
comprising at least a fragment of a lentiviral Gag polypeptide; (B)
a second recombinant yellow fever virus which expresses a second
heterologous polypeptide comprising at least a fragment of a
lentiviral Vif polypeptide; (C) a third recombinant yellow fever
virus which expresses a third heterologous polypeptide comprising
at least a fragment of a lentiviral Nef polypeptide, and (D) a
pharmaceutical carrier, wherein the composition comprises the
mixture in an amount effective to induce a protective or
therapeutic immune response against infection lentiviral.
11. The composition according to claim 10, wherein the Gag
polypeptide is a Gag polypeptide of HIV; the Vif polypeptide is a
Vif polypeptide of HIV; and the Nef polypeptide is a Nef
polypeptide of HIV.
12. A method for inducing a protective or therapeutic immune
response against HIV, wherein the method comprises administering
the composition defined in claim 1 to an individual who needs
it.
13. The method according to claim 12, further comprising
administering an initial dose of the composition of claim 1 in a
priming step prior to administering the composition of claim 1,
wherein the initial dose comprises a DNA encoding one or more of
HIV polypeptides or fragments thereof and/or the initial dosage of
the composition comprises rBCG that expresses one or more of
polypeptides of HIV or fragments thereof.
14. The method according to claim 12, wherein the composition is
administered in two or more subsequent times, waiting for at least
4-12 weeks before the subsequent administration.
15. The method according to claim 12, wherein a protective or
therapeutic immune response against HIV includes a response of CD4+
T cell and/or a response in CD8+ Tcell.
16. A vaccine to induce a protective immune response or a
therapeutic response against HIV, wherein said vaccine comprises:
(A) a recombinant yellow fever virus which expresses a heterologous
polypeptide comprising at least a fragment of a lentiviral
polypeptide; (B) a pharmaceutical carrier; wherein the composition
comprises the recombinant yellow fever virus in an amount effective
to induce a therapeutic or protective immune response against
infection with lentivirus.
17. A vaccine to induce a protective immune response or therapeutic
response against HIV, wherein said vaccine comprises a mixture of
recombinant yellow fever virus, said mixture comprising: (A) a
first recombinant yellow fever virus which expresses a first
heterologous polypeptide comprising at least a fragment of a
lentiviral Gag polypeptide; (B) a second recombinant yellow fever
virus expressing a second heterologous polypeptide comprising at
least a fragment of a lentiviral Vif polypeptide; (C) a third
recombinant yellow fever virus that expresses a third heterologous
polypeptide comprising at least a fragment of a lentiviral Nef
polypeptide; and (D) a pharmaceutical carrier.
18. A 45-269 amino acid fragment of the Gag protein used for
drawing a heterologous cassette of SIV, wherein the fragment
comprises SEQ ID NO:1.
19. A recombinant protein of 45-269 amino acids from Gag protein,
wherein the recombinant protein comprises SEQ ID NO:2.
20. A recombinant gene of Gag 45-269 protein of SIV, wherein the
gene comprises SEQ ID NO:6.
21. A nucleotide sequence of recombinant yellow fever virus which
expresses Gag 45-269 protein, wherein the nucleotide sequence
comprises SEQ ID NO:7.
22. A precursor polyprotein containing a recombinant protein of
amino acids 45-269 from the SIV Gag protein, wherein the precursor
polyprotein comprises SEQ ID NO:8.
23. A method for producing lentivirus proteins or related
polypeptides, wherein said method comprises the steps of: (A)
introducing at least one vector or a vector system in a host cell,
said vector or vector system including a nucleic acid sequence
encoding at least an immunogenic protein of lentivirus or a related
polypeptide, a nucleic acid sequence having at least 90% identity
with sequence SEQ ID NO:6 or SEQ ID NO:7 or an encoded protein
having at least 90% identity with a protein defined by SEQ ID
NOs:1, 2, 8 or 9; (B) expressing the nucleic acid sequence into the
host cell; and (C) producing anthe antigenic protein encoded by
lentivirus or a related polypeptide within the host cell.
24. The method according to claim 23, wherein two or more vectors
or vector systems are introduced.
25. The method according to claim 23, wherein the vector or vector
system includes a nucleic acid sequence encoding two or more
immunogenic proteins of lentiviruses or related polypeptides.
Description
FIELD OF THE INVENTION
[0002] The present invention relates to the development of a
vaccine against lentivirus, and more specifically against primate
lentiviruses such as HIV (Human Immunodeficiency Virus) and SIV
(Simian Immunodeficiency Virus).
[0003] The present invention concerns the use of attenuated
recombinant yellow fever vaccine virus 17D for expression of HIV
and SIV antigens for immunization. The invention provides
immunization schemes using yellow fever virus expressing HIV or SIV
antigens. For example, the recombinant virus may be used in a
"prime-boost" protocol immunizing an individual with a recombinant
polynucleotide or recombinant Bacillus Calmette-Guerin (rBCG)
vaccine, which expresses one or more HIV or SIV antigens, followed
by booster dose with recombinant Yellow Fever virus 17D, which
expresses one or more HIV or SIV antigens.
BACKGROUND OF THE ART
[0004] Over the 28-year period since the scientists identified the
human immunodeficiency virus (HIV) as the cause of the acquired
immunodeficiency syndrome (AIDS), the virus has spread inexorably,
giving rise to one of the most devastating pandemics ever recorded
in history. More than 20 million people have already died of AIDS
and about 33 million people are living with HIV infection.
[0005] However, none of the vaccine regimens tested in HIV vaccine
efficacy trials to date has reduced HIV infection or replication
rates.
[0006] Structural aspects and the enormous variability of the
Envelope glycoprotein have frustrated the efforts to broadly induce
reactive neutralizing antibodies against HIV [1]. Investigators
have therefore focused their attention on T-cell-based vaccines.
The recent trial of a recombinant adenovirus[Ad5]-vectored vaccine
was widely seen as an important test of this concept [3,4].
Unfortunately, vaccinees became infected at higher rates than
control groups [3].
[0007] Several studies have shown the key role of CD+8 T cells in
the control of HIV infections as well as of simian immunodeficiency
virus (SIV) infections. As a result, various modalities of
response-inducing vaccines regarding CD8+ T-cell effector response
have been currently investigated and developed [5, 6, 7, 8].
[0008] Hence, it is also desirable that new approaches are
developed in order to reduce the incidence of HIV infection or
improve the consequences of the infection. As regards this aspect,
vaccines are particularly relevant.
SUMMARY OF THE INVENTION
[0009] The technical matter described herein relates to
pharmaceutical compositions that may be used as immunogenic
compounds or vaccines against lentivirus, and more specifically as
immunogenic compounds or vaccines against HIV (Human
Immunodeficiency Virus) or SIV (Simian Immunodeficiency Virus).
BRIEF DESCRIPTION OF THE FIGURES
[0010] FIG. 1 shows viral growth curves in Vero cells.
[0011] FIG. 2 shows that YF17D replicates and induces neutralizing
antibodies, virus-specific CD8+ T cells, and CD8+ T-cell activation
in rhesus monkeys.
[0012] FIG. 3 shows the study of infection and immunogenicity of
YF17D/SIVGag45-269 virus in rhesus monkeys.
[0013] FIG. 4 shows that vaccination with rYF17D/SIVGag45-269
yielded a robust expansion of Gag-specific responses in a monkey
initially vaccinated with rBCG.
[0014] FIG. 5 shows that the result of the vaccination with
rYF17D/SIVGag45-269 of r01056 [sic].
DETAILED DESCRIPTION OF THE INVENTION
[0015] The described compositions typically include a recombinant
polynucleotide or a panel of recombinant polynucleotides. Adequate
HIV or SIV polynucleotides may encode at least one fragment of a
HIV or SIV polypeptide including Gag, Pol, Rev, Tat, Nef, Vif, Vpx,
Vpr, Env, Vpu, [sic] (preferably at least one fragment of Gag, Vif,
and Nef). The recombinant polynucleotide or panel of recombinant
polynucleotides may be present in a recombinant virus or in a panel
of recombinant viruses which express polypeptides encoded by the
recombinant polynucleotides. The disclosed compositions may be used
in methods for inducing immune response against HIV, which may
include HIV-1 or HIV-2, or SIV (for example, response based on
cytotoxic T cells (CTC), and, optionally, humoral or antibody-based
response. [sic]
[0016] Disclosed compositions may include a mixture of recombinant
polynucleotides, which may be present in a viral vector or in other
nucleotide vector such as a plasmid or a bifunctional vector
("shuttle" vectors). Typically, the polynucleotides are present in
expression vectors to express the polypeptides encoded by the
polynucleotides. Adequate vectors may include but are not limited
to viral vectors (for example, attenuated recombinant viruses). The
pharmaceutical composition may include free recombinant nucleic
acid or recombinant nucleic acid packaged into one or more virus
particles (for example, replication-defective viral particles or
attenuated virus). For instance, the recombinant polynucleotides
contemplated herein may be present in one or more recombinant
viruses (for example, recombinant yellow fever viruses) and the
compositions may include a mixture of recombinant viruses.
[0017] The compositions may include a polynucleotide or a panel of
polynucleotides (for example, at least about 2-9 polynucleotides)
present in one or more expression vectors (for example, a viral
vector or other expression vector, [sic] which encode the complete
Gag polypeptide or a fragment/part of it. Optionally, the
compositions may include a polynucleotide or a panel of
polynucleotides present in one or more expression vectors which
encode the complete Vif polypeptide or a fragment/part of it.
Optionally, the compositions may include a polynucleotide or a
panel of polynucleotides present in one or more expression vectors
which encode the complete Nef polypeptide or a fragment/part of it.
Optionally, the compositions may include a polynucleotide or a
panel of polynucleotides present in one or more expression vectors
which encode the complete Tat polypeptide or a fragment/part of it.
Optionally, the compositions may include a polynucleotide or a
panel of polynucleotides present in one or more expression vectors
which encode Rev polypeptide or a fragment/part of it. Optionally,
the compositions may include a polynucleotide or a panel of
polynucleotides present in one or more expression vectors which
encode the complete Pol polypeptide or a fragment/part of it.
Optionally, the compositions may include a polynucleotide or a
panel of polynucleotides present in one or more expression vectors
which encode the complete Vpr polypeptide or a fragment/part of it.
Optionally, the compositions may include a polynucleotide or a
panel of polynucleotides present in one or more expression vectors
which encode the complete Vpx polypeptide or a fragment/part of it.
The polynucleotides or panels of polynucleotides may encode
different polypeptides or different fragments of polypeptides. The
encoded polypeptides or fragments of polypeptides may overlap (for
example, in about 9-20 amino acids). The fragments are typically in
the length range of 5-100 amino acids (or from 5-50, 10-50, 10-25,
5-15, or 10-15 amino acids) and include at least one epitope of the
polypeptide from which the fragment is derived. In some
embodiments, a fragment may include an N-terminal amino acid which
is not normally present in the complete polypeptide from which the
fragment is derived.
[0018] The pharmaceutical composition may comprise an effective
amount or concentration of recombinant polynucleotides or
recombinant viruses to induce a protective or therapeutic immune
response against HIV infection in humans or SIV infection in
simians. Inducing a protective or therapeutic immune response may
include the potentiation of a CD8+ and/or CD4+ response to one or
more HIV or SIV epitopes. Inducing a protective response may
include the induction of sterilizing immunity against HIV or SIV.
Inducing a therapeutic response may include the reduction of the
viral load of an individual, for example, as determined by the
measurement of the amount of circulating virus before and after
delivering the composition. In some embodiments, the viral load is
reduced to less than about 10,000 vRNA copies/mL (preferably to
less than about 5,000 vRNA copies/mL, more preferably to less than
about 2000 vRNA copies/mL, much more preferably to less than about
1500 vRNA copies/mL, and even more preferably to less than about
1000 vRNA copies/mL) at least about 3 months after the composition
administration. The induction of a therapeutic response may include
the increase in CD4+ T-cell count of the individual (for example,
at least a twofold, threefold, or fourfold increase) at least 3
months after the composition administration.
[0019] Methods inducing protective or therapeutic immune responses
against HIV or SIV infection through the administration of the
pharmaceutical compositions described herein (for example, as
immunogenic compounds or vaccines) to an individual who needs them,
which may include a human who has already acquired or is at risk of
acquiring a lentivirus infection, are also disclosed.
[0020] As used herein, a "nucleic acid vaccine" or "free DNA
vaccine" relates to a vaccine which includes one or more expression
vectors which encode B-cell and/or T-cell epitopes and provide an
immunoprotective response to the individual who is being
vaccinated. Nucleic acid vaccines as defined here, which may
include expression vector plasmids, are not typically encapsidated
into a viral particle. The vaccine nucleic acid is directly
introduced into the cells of the individual who is subjected to the
vaccinal regimen. This approach is described, for example, in U.S.
Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524;
5,679,647; and WO 98/04720. Examples of DNA-based administration
technologies may include pure DNA ("naked DNA") administration,
facilitated distribution (for example, through the use of
bupivacaine, polymers, or peptides), and administration in the
presence of cationic lipid complex or liposomes. Nucleic acids may
be delivered using ballistic administration (for example, as
described in U.S. Pat. No. 5,204,253), by pressure (for example, as
described in U.S. Pat. No. 5,922,687), or by electroporation.
[0021] The nucleic acids used in the compositions and vaccines
disclosed herein may encode one or more viral proteins or portions
of one or more viral proteins. In some embodiments, the nucleic
acids may encode an epitope (for example, a T-cell epitope) which
is not part of a known HIV or SIV functional protein or which is
encoded by a HIV or SIV alternative open reading frame (for
example, a "cryptic" epitope). (See, for example, Maness et al., J.
Exp. Med. (2007), 204:2505-12; and Cardinaud et al., J. Exp. Med.
(2004), 199:1053-1063, which are all incorporated by reference
herein in their entireties). A cryptic epitope may be located in an
open reading frame (ORF) whose translation is not typically
observed in the course of HIV or SIV viral replication. For
example, Maness et al. discloses a cryptic epitope in SIVmac239
called "cRW9", which is located in the +2 reading frame relative to
the ORF encoding the envelope protein. This cryptic epitope is
located in the same ORF that encodes exon 1 of the Rev protein, but
it is downstream of the only known splice donor site and so is not
predicted to be translated under "normal" biological
circumstances.
[0022] The pharmaceutical compositions disclosed herein may be
formulated as vaccines for administration to an individual who
needs them. These compositions may be formulated and/or
administered at dosages and through techniques that are well known
by physicians, taking into account factors such as age, gender,
weight, particular conditions of the patient, and route of
administration. The compositions may include pharmaceutical
carriers, diluents, or excipients as they are known [in the art].
Moreover, the compositions may include preservatives (for example,
antimicrobial or antibacterial agents such as benzalkonium
chloride) or adjuvants.
[0023] The pharmaceutical compositions may be administered
prophylactically or therapeutically. In prophylactic
administration, vaccines may be administered in a sufficient amount
to induce CD8+, CD4+, and/or antibody responses for protection
against infection. In therapeutic applications, vaccines are
administered to a patient in a sufficient amount to induce a
therapeutic effect (for example, CD8+, CD4+, and/or antibody
responses for HIV or SIV antigens or epitopes encoded by the
polynucleotides of the composition [sic], which heals or at least
partially suspends or delays the symptoms and/or complications of
HIV or SIV infection (i.e., a "therapeutically effective
dose").
[0024] The compositions disclosed herein may include recombinant
polynucleotides or recombinant viruses which encode and express
complete HIV or SIV polypeptides or their fragments. As used
herein, a "minigene" encodes only part of a gene. For example, a
minigene for HIV Gag may encode only 5-100 amino acids of the Gag
polypeptide (or 5-50, 10-50, 5-25, 10-25, 5-15, or 10-15 amino
acids). Minigenes which encode only part of a gene are described in
the U.S. patent application Ser. No. 12/022,530, filed on Jan. 30,
2008, which claims the benefit of U.S. Provisional Application No.
60/898,644, filed on Jan. 31, 2007, the contents of which are
incorporated by reference herein in their entireties. For example,
a minigene for HIV Gag may encode a Gag fragment which includes
only 5-100 amino acids of the Gag polypeptide (or 5-50, 10-50,
5-25, 10-25, 5-15, or 10-15 amino acids). In some embodiments, the
recombinant polynucleotide or recombinant viruses may encode and
express a SIV polypeptide fragment as disclosed in Table 1 or a
corresponding HIV polypeptide fragment.
TABLE-US-00001 TABLE 1 Epitope-specific sequences of amino acids.
MHC class I Protein Name Sequence restriction Tat SL8 STPESANL A*01
Nef IW9 IRYPKTFGW B*17 MW9 MHPAQTSQW B*17 YY9 YTSGPGIRY A*02 YY9*
YTYEAYVRY A*02 AL11 ARRHRILDIYL B*08 ML10 MRRSRPSGDL B*08 RL10
RRRLTARGLL B*08 Vif HW8 HLEVQGYW B*17 WY8 WTDVTPNY A*02 RL8
RRDNRRGL B*08 RL9 RRAIRGEQL B*08 Ver KL9 KRLRLIHLL B*08 RL10
RRRWQQLLAL B*08 Pol p10 LV10 LGPHYTPKIV A*01 protease YL8 YHSNVKEL
A*07 Env gp120 CL9 CAPPGYALL A*01 Env gp41 TL9 TVPWPNASL A*01 Env
gp41 RY8 RTLLSRVY A*02 Env gp41 FW9 FHEAVQAVW B*17 Gag p17 GY9
GSENLKSLY A*02 matrix CM9 CTPYDINQM A*01 Gag p27 QI9 QNPIPVGNI A*01
capsid LF8 LAPVPIPF A*01 Gag p27 RW9 RAPRRQGCW B*17 capsid Vpx II11
IPPGNSGEETI A*01
[0025] In some embodiments, the recombinant polynucleotide or
recombinant viruses may comprise a "minigene" of SIV or HIV and
express a SIV polypeptide fragment or a corresponding HIV
polypeptide fragment as disclosed in Tables 2-6.
TABLE-US-00002 TABLE 2 SIV and HIV Gag Protein Fragments Protein
Sequence 1(a) SVLSGKKADELEKIRLRPNGKKKYMLKHVVWAANELDRFGLAESLLE SIV
Gag p17 1(b) SVLSGGKLDKWEKIRLRPGGKKTYQLKHIVWASRELERFAVNPGLLE HIV
Gag p17 2(a) FGLAESLLENKEGCQKILSVLAPLVPTGSENLKSLYNTVDV SIV Gag p17
2(b) FAVNPGLLETGGGCKQILVQLQPSLQTGSEELKSLYNAVAT HIV Gag p17 3(a)
NKEGCQKILSVLAPLVPTGSENLKSLYNTVCVIWCIHAEEKVKHTEEA SIV Gag p17
KQIVQRHLVVETGTTETMPKTSR 3(b)
TGGGCKQILVQLQPSLQTGSEELKSLYNAVATLYCVHQGIEVRDTKEA HIV Gag p17
LDKIEEEQNKSKKKAQQAAA 4(a)
GNYVHLPLSPRTLNAWVKLIEEKKFGAEVVPGFQALSEGCTPYDI SIV Gag p27 4(b)
GQMVHQAISPRTLNAWVKVIEEKAFSPEVIPMFSALSEGATPQDL HIV Gag p24 5(a)
SEGCTPYDINQMLNCVGDHQAAMQIIRDIINEEAADWDLQHPQPAPQQ SIV Gag p27
GQLREPSGSDIAGTTSSVDEQIQWMYRQQNPIP 5(b)
SEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPAHAGPN HIV Gag p24
APGQMREPRGSDIAGTTSTLQEQIGWMTSNPPVP 6(a)
SEGCTPYDINQMLNCVGDHQAAMQIIRDIINEEAADWDLQHPQPAPQQ SIV Gag p27 GQ
6(b) SEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPAHAGPN HIV Gag p24
APGQ 7(a) MYRQQNPIPVGNIYRRWIQLGLQKCVRMYNPTNIPDVKQGPKEPFQSY SIV Gag
p27 VDRFYKSLRAEQTDAAVKNWMTQTLLIQNANPDCKLVLKGLGVNPTLE
EMLTACQGVGGPGQKARLMAEALKEALAPVPIPFAAAQQRGPRKPIKC
WNCGKEGHSARQCRAPRRQGCW 7(b)
MTSNPPVPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYV HIV Gag p24
DRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEE
MMTACQGVGGPSHKARILAEAMSQVTSPANIMMQRGNFRNQRKTIKCF
NCGKEGHLARHCRAPRKKGCW 8(a)
TTSSVDEQIQWMYRQQNPIPVGNIYRRWIQLGLQKCVRMYNPTNIPDV SIV Gag p27
KQGPKEPFQSYVDRFYKSLRAEQTDAAVKNWMTQTLLIQNANPDCKLV
LKGLGVNPTLEEMLTACQGVGGPG 8(b)
TTSTLQEQIGWMTSNPPVPVGEIYKRWIILGLNKIVRMYSPVSILDIR HIV Gag p24
QGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTIL
KALGPAATLEEMMTACQGVGGPS
TABLE-US-00003 TABLE 3 SIV and HIV Vif Protein Fragments 10(a)
MEEEKRWIAVPTWRIPERLERWHSLIKYLKYKTKDLQKVCYVPHFKVGWA SIV Vif
WWTCSRVIFPLQEGSHLEVQGYWHLTPEKGWLSTYAVRITWYSKNFWTDV TPNYADILLH 10(b)
MENRWQVMIVWQVDRMRIKTWKSLVKHHMYISKKAKEWVYRHHYESTHPR HIV Vif
ISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNTQVDP DLADKLIH 11(a)
PNYADILLHSTYFPCFTAGEVRRAIRGEQLLSCCRFPRAHKYQVPSLQYL SIV Vif
ALKVVSDVRSQGENPTWKQWRRDNRRGLRMAKQNSRGDKQRGGKPPTKGA NFPGLAKVLGILA
11(b) PDLADKLIHLHYFDCFSDSAIRHAILGHRVRPKCEYQAGHNKVGSLQYLA HIV Vif
LTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH 12(a)
MEEEKRWIAVPTWRIPERLERWHSLIKYLKYKTKDLQKVCYVPHFKVGWA SIV Vif
WWTCSRVIFP 12(b) MENRWQVMIVWQVDRMRIKTWKSLVKHHMYISKKAKEWVYRHHYESTHPR
HIV Vif ISSEVHIP 13(a)
WWTCSRVIFPLQEGSHLEVQGYWHLTPEKGWLSTYAVRITWYSKNFWTDV SIV Vif
TPNYADILLH 13(b) ESTHPRISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRY
HIV Vif NTQVDPDLADKLIH 14(a)
PNYADILLHSTYFPCFTAGEVRRAIRGEQLLSCCRFPRAHKYQVPSLQYL SIV Vif
ALKVVSDVR 14(b) PDLADKLIHLHYFDCFSDSAIRHAILGHRVRPKCEYQAGHNKVGSLQYLA
HIV Vif LTALITPKK 15(a)
LALKVVSDVRSQGENPTWKQWRRDNRRGLRMAKQNSRGDKQRGGKPPTKG SIV Vif
ANFPGLAKVLGILA 15(b) LALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH
HIV Vif
TABLE-US-00004 TABLE 4 SIV and HIV Nef Protein Fragments 16(a)
GLDKGLSSLSCEGQKYNQGQYMNTPWRNPAEEREKLAYRKQNMDDIDEED SIV Nef
DDLVGVSVRPKVPLRTMSYKLAIDMSHFIKEKGGLEGIYYSARRHRILDI
YLEKEEGIIPDWQDYTSGPGIRYPKTFGWLWKLVPVNVSDEAQEDEEHYL MHPAQTSQWDDPWGEV
16(b) MGGKWSKSSKAGWPQVREKIKQTPPAAEGVGAVSQDLDKHGAITSSNMNN HIV Nef
ADCVWLQAQEDEEVGFPVRPQVPLRPMTFKGAFDLSFFLKEKGGLDGLIY
SRKRQEILDLWVYNTQGFFPDWQNYTPGPGVRLPLCFGWCFKLVPVDPRE
VEEDNKGENNCLLHPLSQHGMEDEHKEV 17(a)
GLDKGLSSLSCEGQKYNQGQYMNTPWRNPAEEREKLAYRKQNMDDIDEED SIV Nef DD 17(b)
MGGKWSKSSKAGWPQVREKIKQTPPAAEGVGAVSQDLDKHGAITSSNMNN HIV Nef
ADCVWLQAQEDEE 18(a)
DDIDEEDDDLVGVSVRPKVPLRTMSYKLAIDMSHFIKEKGGLEGIYYSAR SIV Nef RH 18(b)
DCVWLQAQEDEEVGFPVRPQVPLRPMTFKGAFDLSFFLKEKGGLDGLIYS HIV Nef RKR
19(a) GIYYSARRHRILDIYLEKEEGIIPDWQDYTSGPGIRYPKTFGWLWKLVPV SIV Nef
19(b) GLIYSRKRQEILDLWVYNTQGFFPDWQNYTPGPGVRLPLCFGWCFKLVPV HIV Nef
20(a) GWLWKLVPVNVSDEAQEDEEHYLMHPAQTSQWDDPWGEV SIV Nef 20(b)
GWCFKLVPVDPREVEEDNKGENNCLLHPLSQHGMEDEHKEV HIV Nef
TABLE-US-00005 TABLE 5 SIV and HIV Tat Protein Fragments 21(a)
METPLREQENSLESSNERSSCISEADASTPESANLGEEILSQLYRPLEA SIV Tat 21(b)
MEPVDPRLEPWKHPGSQPKTPCTKCYCKKCCLHCQVCFMTKGLGISYGRK HIV Tat 22(a)
SQLYRPLEACYNTCYCKKCCYHCQFCFLKKGLGICYEQSRKRRRTPKKAK SIV Tat 22(b)
SQPKTPCTKCYCKKCCLHCQVCFMTKGLGISYGRKKRRQRRRAPQDNKNH HIV Tat
TABLE-US-00006 TABLE 6 SIV and HIV Rev Protein Fragments 23(a)
MSNHEREEELRKRLRLIHLLHQTNPYPTGPGTANQRRQRKRRWRRRWQQ SIV Rev 23(b)
MAGRSGSTDEELLRAVRIIKILYQSNPYPSSEGTRQARRNRRRRWRARQR HIV Rev 24(a)
RRWRRRWQQLLALADRIYSFPDPPTDTPLDLAIQQLQNLAIESIPDPPTN SIV Rev
TPEALCDPTEDSRSPQD 24(b)
RRNRRRRWRARQREICALSERILSSCLGRPTEPVPLPLPPLERLTLDCSE HIV Rev
DCGTSGTQQSQGTETGVGRPQISGE
[0026] A minigene of SIV or a minigene of HIV may encode a
polypeptide comprising a SIV or HIV polypeptide fragment and also
comprise an N-terminal methionine. A minigene of SIV or a minigene
of HIV may include codon sequences which are optimized for
expression (for example, in animal, organism or recombinant virus
as contemplated herein). Exemplary SIV minigenes including codon
sequences which are optimized for expression by the yellow fever
virus are depicted in Table 7.
TABLE-US-00007 TABLE 7 Optimized-Sequence Minigenes Name 1.
SIVmac239Gag(6-52) DNA Seq. 5'-
GTCGACAGGAGCCACCATGAGCGTGCTGAGCGGCAAGAAGGCCGACGAGC
TGGAGAAGATCCGGCTGCGGCCCAACGGCAAGAAGAAATACATGCTGAAG
CACGTGGTGTGGGCCGCCAACGAGCTGGACCGGTTCGGCCTGGCCGAGAG
CCTGCTGGAGTGAGAATTCGCGGCCGC-3' AA Seq.
MSVLSGKKADELEKIRLRPNGKKKYMLKHVVWAANELDRFGLAESLLE 2.
SIVmac239Gag(44-84) DNA Seq. 5'-
GTCGACAGGAGCCACCATGTTCGGCCTGGCCGAGAGCCTGCTGGAGAACA
AGGAGGGCTGCCAGAAGATCCTGAGCGTGCTGGCCCCTCTGGTGCCCACC
GGCAGCGAGAACCTGAAGAGCCTGTACAACACCGTGTGCGTGTGAGAATT CGCGGCCGC-3' AA
Seq. MFGLAESLLENKEGCQKILSVLAPLVPTGSENLKSLYNTVCV 3.
SIVmac239Gag(76-123) DNA Seq. 5'-
GTCGACAGGAGCCACCATGAAGAGCCTGTACAACACCGTGTGCGTGATCT
GGTGCATCCACGCCGAGGAGAAGGTGAAGCACACCGAGGAGGCCAAGCAG
ATCGTGCAGCGGCACCTGGTGGTGGAGACCGGCACCACCGAGACCATGCC
CAAGACCAGCCGCTGAGAATTCGCGGCCGC-3' AA Seq.
MKSLYNTVCVIWCIHAEEKVKHTEEAKQIVQRHLVVETGTTETMPKTSR 4.
SIVmac239Gag(142-186) DNA Seq. 5'-
GTCGACAGGAGCCACCATGGGCAACTACGTGCACCTGCCCCTGAGCCCCC
GGACCCTGAACGCCTGGGTGAAGCTGATCGAGGAGAAGAAGTTCGGCGCC
GAGGTGGTGCCCGGCTTCCAAGCCCTGAGCGAGGGCTGCACCCCCTACGA
CATCTGAGAATTCGCGGCCGC-3' AA Seq.
MGNYVHLPLSPRTLNAWVKLIEEKKFGAEVVPGFQALSEGCTPYDI 5.
SIVmac239Gag(178-258) DNA Seq. 5'-
GTCGACAGGAGCCACCATGAGCGAGGGCTGCACCCCCTACGACATCAACC
AGATGCTGAACTGCGTGGGCGACCACCAGGCCGCCATGCAGATCATCCGG
GACATCATCAACGAGGAGGCCGCCGACTGGGACCTGCAGCACCCCCAGCC
CGCCCCCCAGCAAGGCCAGCTGCGGGAGCCCAGCGGCAGCGACATCGCCG
GCACCACCAGCAGCGTGGACGAGCAGATCCAGTGGATGTACCGGCAGCAG
AACCCCATCCCCTGAGAATTCGCGGCCGC-3' AA Seq.
MSEGCTPYDINQMLNCVGDHQAAMQIIRDIINEEAADWDLQHPQPAPQQG
QLREPSGSDIAGTTSSVDEQIQWMYRQQNPIP 6. SIVmac239Gag(250-415) DNA Seq.
5'- GTCGACAGGAGCCACCATGTACCGGCAGCAGAACCCCATCCCCGTGGGCA
ACATCTACCGGCGGTGGATCCAGCTGGGCCTGCAGAAATGCGTGCGGATG
TACAACCCCACCAACATCCTGGACGTGAAGCAGGGCCCCAAGGAGCCCTT
CCAGAGCTACGTGGACCGGTTCTACAAGAGCCTGCGGGCCGAGCAGACCG
ACGCCGCCGTGAAGAACTGGATGACCCAGACCCTGCTGATCCAGAACGCC
AACCCCGACTGCAAGCTGGTGCTGAAGGGCCTGGGCGTGAACCCCACCCT
GGAGGAGATGCTGACCGCCTGCCAGGGCGTGGGCGGCCCCGGCCAGAAGG
CCCGGCTGATGGCCGAGGCCCTGAAGGAGGCCCTGGCCCCCGTGCCCATC
CCCTTCGCCGCCGCCCAGCAGCGGGGCCCCCGGAAGCCCATCAAGTGCTG
GAACTGCGGCAAGGAGGGCCACAGCGCCCGGCAGTGCCGGGCCCCCCGGC
GGCAGGGCTGCTGGTGAGAGAATTCGCGGCCGC-3' AA Seq.
MYRQQNPIPVGNIYRRWIQLGLQKCVRMYNPTNILDVKQGPKEPFQSYVD
RFYKSLRAEQTDAAVKNWMTQTLLIQNANPDCKLVLKGLGVNPTLEEMLT
ACQGVGGPGQKARLMAEALKEALAPVPIPFAAAQQRGPRKPIKCWNCGKE GHSARQCRAPRRQGCW
7. SIVmac239Vif(1-110) DNA Seq. 5'-
GTCGACAGGAGCCACCATGGAGGAGGAGAAGCGGTGGATCGCCGTGCCCA
CCTGGCGGATCCCCGAGCGGCTGGAGCGGTGGCACAGCCTGATCAAGTAC
CTGAAGTACAAGACCAAGGACCTGCAGAAGGTGTGCTACGTGCCCCACTT
CAAAGTGGGCTGGGCCTGGTGGACCTGCAGCCGGGTGATCTTCCCCCTGC
AAGAGGGCAGCCACCTGGAGGTGCAGGGCTACTGGCACCTGACCCCCGAG
AAGGGCTGGCTGAGCACCTACGCCGTGCGGATCACCTGGTACAGCAAGAA
CTTCTGGACCGACGTGACCCCCAACTACGCCGACATCCTGCTGCACTGAG AATTCGCGGCCGC-3'
AA Seq. MEEEKRWIAVPTWRIPERLERWHSLIKYLKYKTKDLQKVCYVPHFKVGWA
WWTCSRVIFPLQEGSHLEVQGYWHLTPEKGWLSTYAVRITWYSKNFWTDV TPNYADILLH 8.
SIVmac239Vif(102-214) DNA Seq. 5'-
GTCGACAGGAGCCACCATGCCCAACTACGCCGACATCCTGCTGCACAGCA
CCTACTTCCCCTGCTTCACCGCCGGCGAGGTCCGGCGGGCCATCCGGGGC
GAGCAGCTGCTGAGCTGCTGCCGGTTCCCCCGGGCCCACAAGTACCAGGT
GCCCAGCCTGCAGTACCTGGCCCTGAAGGTGGTGAGCGACGTGCGGAGCC
AGGGCGAGAACCCCACCTGGAAGCAGTGGCGGCGGGACAACCGGCGGGGC
CTGCGGATGGCCAAGCAGAACAGCCGGGGCGACAAGCAGCGGGGCGGCAA
GCCTCCCACCAAGGGCGCCAACTTCCCCGGCCTGGCCAAGGTGCTGGGCA
TCCTGGCCTGAGAATTCGCGGCCGC-3' AA Seq.
MPNYADILLHSTYFPCFTAGEVRRAIRGEQLLSCCRFPRAHKYQVPSLQY
LALKVVSDVRSQGENPTWKQWRRDNRRGLRMAKQNSRGDKQRGGKPPTKG ANFPGLAKVLGILA
9. SIVmac239Nef(45-210) DNA Seq. 5'-
GTCGACAGGAGCCACCATGGGCCTGGACAAGGGCCTGAGCAGCCTGAGCT
GCGAGGGCCAGAAGTACAACCAGGGCCAGTACATGAACACCCCCTGGCGG
AACCCCGCCGAGGAGCGGGAGAAGCTGGCCTACCGGAAGCAGAACATGGA
CGACATCGACGAGGAGGACGACGACCTGGTGGGCGTGAGCGTGCGGCCCA
AGGTGCCCCTGCGGACCATGAGCTACAAGCTGGCCATCGACATGAGCCAC
TTCATCAAGGAGAAGGGCGGCCTGGAGGGCATCTACTACAGCGCCCGGCG
GCACCGGATCCTGGACATCTACCTGGAGAAGGAGGAGGGCATCATCCCCG
ACTGGCAGGACTACACCAGCGGCCCCGGCATCCGGTACCCCAAGACCTTC
GGCTGGCTGTGGAAGCTGGTGCCCGTGAACGTGAGCGACGAGGCCCAGGA
GGACGAGGAGCACTACCTGATGCACCCCGCCCAGACCAGCCAGTGGGACG
ACCCCTGGGGCGAGGTGTGAGAATTCGCGGCCGC-3' AA Seq.
MGLDKGLSSLSCEGQKYNQGQYMNTPWRNPAEEREKLAYRKQNMDDIDEE
DDDLVGVSVRPKVPLRTMSYKLAIDMSHFIKEKGGLEGIYYSARRHRILD
IYLEKEEGIIPDWQDYTSGPGIRYPKTFGWLWKLVPVNVSDEAQEDEEHY
LMHPAQTSQWDDPWGEV
[0027] The viral vectors, recombinant bacteria, and nucleic acids
used in the compounds and vaccines disclosed herein may encode one
or more proteins of HIV or SIV or portions of one or more proteins
of HIV or SIV. In some embodiments, nucleic acids may encode an
epitope (for example, a T-cell epitope) which is not part of a
known HIV or SIV functional protein or which is encoded by a HIV or
SIV alternative open reading frame (i.e., a "cryptic" epitope).
(See, for example, Maness et al., J. Exp. Med. (2007), 204:2505-12;
and Cardinau et al., J. Exp. Med. (2004), 199:1053-1063, which are
all incorporated by reference herein in their entireties). A
cryptic epitope may be located in an open reading frame (ORF) whose
translation is not typically observed in the course of HIV or SIV
viral replication. For example, Maness et al. discloses a cryptic
epitope in SIVmac239 called "cRW9", which is located in the +2
reading frame relative to the ORF encoding the envelope protein.
This cryptic epitope is located in the same ORF that encodes exon 1
of the Rev protein, but it is at the 3' side ("downstream") of the
donor processing site ("donor splicing site") and so its
translation would not be expected under "normal" biological
conditions.
[0028] Any of the conventional vectors used for expression in
eukaryotic cells may be used to directly immunize an individual
with nucleic acid. Expression vectors containing regulatory
elements of eukaryotic viruses may be used in eukaryotic expression
vectors (for example, vectors containing SV40, CMV, or retroviral
enhancers or promoters). Exemplary vectors include vectors
expressing proteins under the direction of such promoters as SV40
early promoter, SV40 late promoter, metallothionein promoter, human
cytomegalovirus promoter, murine mammary tumor virus promoter, and
Rous sarcoma virus promoter. Industrial amounts for therapeutic and
prophylactic use of plasmid DNA may be yielded, for example, by
fermentation in E. coli, followed by purification. Aliquots from
the working cell bank are used to inoculate a culture medium, and
cultured until saturation in shake flasks or in a bioreactor
according to widely known techniques. Plasmid DNA may be purified
by standard bioseparation technologies such as solid phase ion
exchange resins. If required, the DNA may be isolated from the
open-circular and linear forms using gel electrophoresis or other
methods.
[0029] The purified plasmid DNA may be prepared for injection using
a wide range of formulations, such as, for example, lyophilized DNA
which may be reconstituted in phosphate-buffered sterile saline
(PBS) solution. The purified DNA may be introduced into an
individual through any appropriate method; for example, by
intramuscular (IM) or intradermal (ID) administration.
[0030] In order to maximize the immunotherapy effects of DNA
vaccines, alternative methods may be desirable to formulate
purified plasmid DNA. A variety of methods has been described, and
new techniques may become available. For example, cationic lipids
may be used in the formulation of the pharmaceutical composition
disclosed herein (for example, as described in WO 93/24640; U.S.
Pat. No. 5,279,833; and WO 91/06309). Protective, interactive,
noncondensing compounds (PINC) may be used in the formulation of
the pharmaceutical composition disclosed herein (for example,
glycolipids, fusogenic liposomes, and peptides).
[0031] The term "vector" refers to a few means by which nucleic
acid may be introduced into a host organism or tissue. There are
several types of vectors, including viruses, plasmids,
bacteriophages, cosmids, and bacteria. As used herein, a
"recombinant virus" or "viral vector" relates to the recombinant
viral nucleic acid which has been genetically modified to express a
heterologous polypeptide (for example, a HIV polypeptide or a
fragment thereof). The recombinant viral nucleic acid typically
includes cis-acting elements for the expression of the heterologous
polypeptide. Typically, the recombinant viral nucleic acid can be
packaged into a virus which is able to infect a host cell. For
example, the recombinant viral nucleic acid may include cis-acting
elements for packaging. Generally, the viral vector is not able to
replicate or is attenuated. An "attenuated recombinant virus"
refers to a virus which has been genetically modified by modern
molecular biological methods, making it less virulent than the wild
type, usually by mutation or deletion of specific genes. For
example, the recombinant viral nucleic acid may not have an
essential gene for an efficient production or for the production of
the infectious virus.
[0032] The recombinant viral nucleic acid may function as a vector
for an immunogenic retroviral protein in view of the fact that the
recombinant viral nucleic acid comprises exogenous nucleic acid.
The recombinant virus may be introduced in a vaccinated human by
standard methods for vaccination using live vaccines. A live
vaccine as contemplated herein may be administered using, for
example, from about 10.sup.4 to 10.sup.8 units of viral
particles/dose, or 10.sup.6 to 10.sup.9 pfu/dose. The actual
dosages of this vaccine may be readily determined by any expert in
the vaccine field.
[0033] Numerous virus species may be used as recombinant virus
vectors for the pharmaceutical composition disclosed herein.
Preferred recombinant virus for a viral vaccine is the yellow fever
vaccine virus (for example, 17-D or similar).
[0034] In some embodiments, attenuated recombinant bacteria or
mycobacteria may be used as vectors in the pharmaceutical
compositions and vaccines disclosed herein (for example, Bacillus,
Shigella, Salmonella, Listeria or Yersinia, attenuated recombinant
bacteria). Recombinant bacterial vaccine vectors are described by
Daudel et al., "Use of attenuated bacteria as delivery vectors for
DNA vaccines", Expert Review of Vaccines, Volume 6, Number 1,
February, 2007, pp. 97-110(14); Shata et al., "Recent advances with
recombinant bacterial vaccine vectors", Molec. Med. Today (2000),
Volume 6, Number 2, Feb. 1, 2000, pages 66-71; Clare & Dougan,
"Live Recombinant Bacterial Vaccines", Novel Vaccination
Strategies, Apr. 16, 2004 (Editor Stefan H. E. Kaufman); Gentaschev
et al., "Recombinant Attenuated Bacteria for the Delivery of
Subunit Vaccines", Vaccine, Volume 19, Numbers 17-19, Mar. 21,
2001, pages 2621-2628; Garmory et al., "The Use of live attenuated
bacteria as a delivery system for heterologous antigens", J. Drug
Target. 2003; 11 (8-10): 471-9; U.S. Pat. No. 6,383,496; and U.S.
Pat. No. 6,923,958 (which are all incorporated by reference herein
in their entireties).
[0035] In some embodiments, the recombinant bacterium or
mycobacterium is the recombinant Bacillus Calmette-Guerin (or
Bacillus Calmette-Guerin, BCG). The BCG is an attenuated strain of
the live bovine tuberculosis bacillus, Mycobacterium bovis, which
lost its virulence in humans by being specially cultured in an
artificial medium over years. The use of a recombinant BCG (rBCG)
expressing the codon-optimized HIV-1 Gag for mice immunization in a
"prime-boost" regimen has been described. Promkhatkaaew D. et al.,
"Prime-boost immunization of codon optimized HIV-1 CRFO1 AE Gag in
BCG with recombinant vaccinia virus elicits MHC class I and II
immune responses in mice", Immunol Invest. 2009; 38(8):762-79.
[0036] The compositions included in the vaccine regimen as
contemplated herein may be coadministered or administered
sequentially with another immunological antigenic vaccine or in
therapeutic compositions, including an adjuvant, a biological or
chemical agent given in combination with or genetically fused to an
antigen to increase the antigen immunogenicity. Additional
therapeutic agents may include but are not limited to CD40 or
interleukin-2 (IL-2) ligand in a sufficient amount to further
potentiate CD8+ T cell, CD4+ T cell, and antibody responses. These
other compositions may also include purified antigens of the
immunodeficiency virus or the expression of such antigens by a
second recombinant vector system which can give rise to additional
therapeutic compositions.
[0037] The pharmaceutical composition disclosed herein may be
delivered by various routes. The typical delivery routes include
parenteral administration (for example, intradermal, intramuscular,
or subcutaneous delivery). Other routes include oral, intranasal,
intravaginal, and intrarectal administration. The pharmaceutical
composition may be formulated for intranasal or pulmonary delivery.
The pharmaceutical composition formulations may include liquid
formulations (for example, for oral, nasal, anal, or vaginal
administration, among others, including suspensions, syrups, or
elixirs) and preparations for parenteral, subcutaneous,
intradermal, intramuscular, or intravenous administration (for
example, injectable administration) such as sterile suspensions or
emulsions.
[0038] As used herein, the term "adjuvant" refers to a compound or
mixture which increases the immune response to an antigen. An
adjuvant may serve as a tissue depot that slowly releases the
antigen and also as a lymphoid system activator which
nonspecifically amplifies the immune response. Examples of
adjuvants which may be employed include the adjuvant MPL-TDM
(Monophosphoryl lipid A/synthetic trehalose dicorynomycolate; for
example, available from GSK Biologicals). Another appropriate
adjuvant is the AS021/AS02 immunostimulatory adjuvant (GSK). These
immunostimulatory adjuvants are formulated to promote an intense
T-cell response and include QS-21, a saponin from Quillay
saponaria, the TL4 ligand, a monophosphoryl lipid A, together in a
lipid or liposomal carrier. Other adjuvants include but are not
limited to nonionic block copolymer adjuvants (for example,
CRL1005), aluminum phosphates (for example, A1PO4), R-848 (a
Th1-like adjuvant), imiquimod, PAM3CYS, poly (I:C), loxoribine,
potentially useful human adjuvants such as BCG (Bacillus
Calmette-Guerin) and Corynebacterium parvum, CpG
oligodeoxynucleotides (ODN), antigens derived from cholera toxin
(for example, CTA1-DD), lipopolysaccharide adjuvants, complete
Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral
gels such as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil or
hydrocarbon emulsions in water (for example, MF59 available from
Novartis Vaccines or Montanide ISA 720), keyhole-limpet
hemocyanins, and dinitrophenol.
[0039] The pharmaceutical compositions disclosed herein may also or
additionally comprise at least one antiviral chemotherapy compound.
Nonlimiting examples may be selected from at least one compound of
the group consisting of gamma globulin, amantadine, guanidine,
benzimidazole hydroxide, interferons, interleukin-16,
thiosemicarbazones, methisazones, rifampicin, ribavirin, pyrimidine
analogs (for example, AZT and/or 3TC), purine analogs, foscarnet,
phosphonoacetic acid, acyclovir, dideoxynucleosides, protease
inhibitors (for example, saquinavir; indinavir; ritonavir; AG 1343;
and VX-2/78), chemokines, such as RANTES, MIP1x or MIP1b, or
ganciclovir.
[0040] As used herein, a "prime-boost vaccination regimen" refers
to a regimen in which a first composition is administered to an
individual one or more times (for example, two or three times with
about 2, 3, or 4 weeks between administrations), and, after a
certain period of time (for example, about two weeks, about four
weeks, about two months, about three months, about four months,
about five months, about six months, or more), a second composition
is administered to the individual, which may be equal to or
different from the first composition. The second composition may
also be administered more than once, with at least 2, 3, or 4 weeks
between administrations. In some embodiments, the individual is
given the first composition, which comprises the recombinant yellow
fever virus 17D expressing one or more HIV or SIV polypeptides, or
their fragments, or rBCG expressing one or more HIV or SIV
polypeptides, or their fragments. Subsequently, the individual
receives the second composition, which comprises the recombinant
yellow fever virus 17D expressing one or more HIV or SIV
polypeptides, or their fragments, DNA encoding one or more HIV or
SIV polypeptides, or their fragments, or rBCG expressing one or
more HIV or SIV polypeptides or their fragments. The first and the
second compositions may be the same or different.
[0041] The pharmaceutical compositions disclosed herein may be
delivered to individuals at risk of developing HIV or SIV infection
or to individuals who are already infected with HIV or SIV. To
evaluate the efficacy of the vaccine, the immune response may be
assessed by measuring the induction of CD8+ and antibody responses
for particular epitopes. CD8+ T-cell responses may be measured, for
example, by using cultured or fresh PBMC tetramer staining, ELISPOT
assays, or functional cytotoxicity assays, which are well known
among experts, and are described herein. Antibody responses may be
measured by widely known assays such as ELISA. Determinations of
viral titers or loads and CD4+ T cells after immunization may be
performed in individuals who have already been infected. For
example, individuals may present a decline in the viral load and an
increase in the CD4+cell count after immunization (for example, at
least a twofold, threefold, or fourfold increase in the CD4+cell
count in relation to the count obtained prior to immunization).
[0042] The invention will now be described in detail regarding
certain embodiments of the invention.
[0043] In some embodiments of the recombinant viruses contemplated
herein, the virus is a yellow fever vaccinal virus engineered to
express a heterologous polypeptide. The heterologous polypeptide
may include a HIV polypeptide (HIV-1 or HIV-2), a SIV polypeptide,
or a fragment of them (for example, a fragment consisting of at
least 8, 9, 10, 20, 30, 40, 50, 100, or 200 contiguous amino acids
of a HIV Gag polypeptide or a SIV Gag polypeptide). In other
embodiments, the heterologous polypeptide is a polypeptide with at
least 90%, 95%, 96%, 97%, 98%, or 99% of sequence identity with a
HIV polypeptide, a SIV polypeptide, or a fragment of them (for
example, a fragment consisting of at least 8, 9, 10, 20, 30, 40,
50, 100, or 200 contiguous amino acids of a HIV Gag polypeptide or
a SIV Gag polypeptide). Sequences for HIV-1 strains and several
genes of HIV-1 strains (including the gene for Gag polypeptide) are
publicly available in GenBank; for example, the sequences available
under accession numbers: NC.sub.--001802; EU293448, EU293447,
EU293446, EU293445, EU293444, FJ195091, FJ195090, FJ195089,
FJ195088, FJ195087, FJ195086, EU884501, EU786681, EU786680,
EU786679, EU786678, EU786677, EU786676, EU786675, EU786674,
EU786673, EU786672, EU786671, EU786670, EU697909, EU697908,
EU697907, EU697906, EU697905, EU697904, U71182, EU69324, EU861977,
FJ213783, FJ213782, FJ213781, FJ213780, AB428562, AB428561,
AB428560, AB428559, AB428558, AB428557, AB428556, AB428555,
AB428554, AB428553, AB428552, AB428551, DQ295195, DQ295196,
DQ295194, DQ295193, DQ295192, EU446022, EU735540, EU735539,
EU735538, EU735537, EU735536, EU735535, EU110097, EU110096,
EU110095, EU110094, EU110093, EU110092, EU110091, EU110090,
EU110089, EU110088, EU110087, EU110086, EU110085, AF193277,
AF193276, AF049337, EU541617, EU220698, EF469243, DQ912823,
DQ912822, EU000516, EU000515, EU000514, EU000513, EU000512,
EU000511, EU000510, EU000509, EU000508, EU000507, and EU884500;
whose submissions to GenBank are incorporated by reference herein
in their entireties. Primary and field isolates of HIV are
available from the National Institute of Allergy and Infectious
Diseases (NIAID), which has contracted the services of Quality
Biological (Gaithersburg, Md.) to make these strains available.
Strains are also available from the World Health Organization
(WHO), Geneva, Switzerland. In a preferred embodiment, the selected
polypeptide has a consensus sequence as determined by the
comparison of three or more HIV strains. As disclosed herein, the
polynucleotides encoding a HIV polypeptide or a SIV polypeptide may
be "optimized" for expression in a human cellular environment or
simian cellular environment, respectively. Thus, the use of
polynucleotides specifically including HIV genes that are
codon-optimized for expression in a human cellular environment is
contemplated herein. Optimized synthetic HIV gag genes are
described in U.S. Pat. No. 6,696,291.
[0044] The recombinant yellow fever viruses contemplated herein are
prepared through the insertion of the sequence encoding the
heterologous polypeptide into the genome of the yellow fever virus
or vector which can replicate the yellow fever virus. Adequate
yellow fever viruses include attenuated strains such as YF17D.
Adequate insertion sites for the sequence encoding the heterologous
polypeptide include sites between the sequences encoding viral
proteins E and NS1. The sequence encoding the heterologous
polypeptide may be codon-optimized based on the use of the codon
for the yellow fever virus genome.
[0045] In another embodiment of the recombinant viruses described
herein, the virus is a genetically modified vaccinal YF 17D virus
which expresses amino acids 45-269 of SIVmac239Gag
(rYF17D/SIVGag45-269), and is prepared through the insertion of a
codon-optimized sequence based on YF genome between the genes which
encode viral proteins E and NS1.
[0046] In one example, the antigen used for expression by the
yellow fever 17D virus was a fragment of a Gag protein comprising
amino acid residues 45 to 269. Gag is a precursory protein that, on
being processed by the viral protease, generates the internal
structural proteins of the mature virion. Gag protein induces a
strong protective immune response mediated by CD8+ T cells in
rhesus monkeys and, on a smaller scale, in humans. This immune
response is very important since it may provide protection against
viral infection or reduce the viral load and delay the onset of
disease.
[0047] In some embodiments, the following criteria may be used to
select a Gag fragment to be cloned and expressed in the E/NS1
region of 17D virus genome. First, the presence of CD8+ T-cell
epitopes in the Gag polypeptide may be taken into consideration
based on immunological studies in rhesus monkeys. Furthermore,
preferably, the insertion of the Gag polypeptide should not disturb
the translocation of the YF precursory protein into the endoplasmic
reticulum, as well as its processing by viral and cell proteases,
if these steps occur during virus morphogenesis and assembly. Thus,
preferably, the Gag polypeptide fragment should not be hydrophobic
or contain a transmembrane motif, which might produce a rupture or
significantly destabilize the correct topology of the membrane and
precursory protein processing. Therefore, the selected Gag
polypeptide fragment is hydrophilic and does not include apparent
transmembrane domains. Besides, a very flexible motif, KESSIG, is
fused to the C-terminal of the Gag sequence. This sort of motif is
present in all flaviviruses and proteins binding domain III and the
anchorage region ("stem anchor") of E protein. It is a very
flexible motif and was used here as a separating sequence to
connect and move away the Gag fragment and alpha helices of the
anchorage domain ("stem anchor") of truncated dengue 4 virus E
protein in the recombinant cassette.
[0048] Moreover, the virus vaccines presently disclosed differ from
the virus vaccines of the state of the art by using an attenuated
vector structure and emphasizing the generation of cell immune
responses for specific viral epitopes in certain proteins. The
strategy for vector virus replication is maximizing the production
of antigens in an appropriate cell location to induce an
appropriate response in terms of specificity and magnitude.
[0049] Vaccination with a single dose of attenuated yellow fever
virus 17D causes a limited viral infection and promotes the
generation of neutralizing antibodies as well as T-cell responses
which confer protective immunity to more than 95% of vaccinees,
providing immunity for over 30 years. The yellow fever 17D vaccine
is regarded as one of the most successful vaccines available.
Vaccine production concerns a well-established process and, since
1937, over 540 million doses have been used in humans with a
minimal incidence of severe side effects [9,10].
[0050] The current methods are based on the methodology that was
previously developed by two of the present inventors. The
methodology is described in WO07051267, which is incorporated by
reference herein.
[0051] The method for the production of recombinant viruses with
nucleotide sequences totally or partially encoding the heterologous
proteins, as described in WO07051267, may comprise the following
steps:
[0052] (a) modification of the heterologous nucleotide sequences in
such a way that, when they are cloned in the vector virus, they
present, in the 5' region, nucleotides of the 5' end of the vector
virus NS1 gene or functionally equivalent sequences, and, in its 3'
end, the totality or part of the anchorage genome region ("stem
anchor") of E protein of the vector virus or functionally
equivalent sequences, which do not compromise the structure and
replication of said vector virus;
[0053] (b) insertion of the modified heterologous sequences as
described in (a) into the intergenic region between the genes
encoding E structural protein and NS1 nonstructural protein of the
vector virus;
[0054] (c) regeneration of the nonpathogenic recombinant virus,
having the heterologous sequences stably integrated into the viral
genome at the insertion point described in (b), and expression of
the heterologous sequence in such a way that the corresponding
protein induces an appropriate immune response.
[0055] Thus, the inventors found that the strategy for E/NS1
insertion may be applied to generate recombinant vaccines against
lentiviruses, particularly the HIV (Human Immunodeficiency
Virus).
In one embodiment of a recombinant virus as contemplated herein, an
attenuated recombinant virus is a yellow fever vaccinal virus 17D
expressing a sequence of SIVmac239 Gag sequences which is used as a
viral vector to originate SIV-specific CD8+ T-cell responses in
rhesus monkeys. The model of Asian rhesus monkey was employed to
validate the expression of HIV antigens in the yellow fever 17D
virus vector because the SIV-infected rhesus monkey model
reproduces, in many aspects, the immunodeficiency that occurs in
HIV-infected humans. First, several SIV strains are closely related
to HIV-1 strains and infect CD4+ T cells, giving rise to a decline
in their counts over time. SIV infection causes a disease that is
similar to AIDS in most of the monkeys infected for one year after
inoculation. Moreover, many genes of the monkeys which encode key
proteins in the immune system are similar to those of humans.
Equivalents of the genes for HLA class, class II [sic] and T-cell
receptor (TCR) are found in the monkeys. Orthologs of HLA-A, -B, -E
and -F locations were isolated in rhesus monkeys. In both
infections, CD8+ T cells select the major part of the sequence
variation outside the envelope protein and the natural control of
viral replication is associated with certain MHC-I alleles
[11,12].
[0056] Consequently, as disclosed herein, the vaccination of rhesus
monkeys with different formulations containing recombinant yellow
fever virus was assessed so as to determine the potential use of
17D vector in the development of new vaccines against HIV. The
recombinant yellow fever virus obtained expresses a SIVmac239 Gag
fragment encompassing amino acids 45 to 269. Gag is a precursory
protein and, in the course of viral replication, it undergoes
proteolytic splitting by the viral protease, producing the internal
structural proteins of the mature virion. It was determined that
multiple Gag-specific responses are important in the control of SIV
replication. This may be due to infected cells which present
Gag-derived CD8+ T-cell epitopes right after the viral entry.
Gag-specific CD8+ T cells recognize infected cells in up to 2 hours
after the infection. Six hours after the infection, Gag-specific
CD8+ T cells eliminate the infected cells, both before and after
the integration of proviral DNA and viral protein synthesis. These
findings demonstrate that Gag-specific CD8+ T cells do not require
productive cell infection or even protein synthesis "again" to
recognize the infected cells and eliminate them. This suggests that
Gag-specific CD8+ T cells may be much more important than
previously imagined.
[0057] In order to reach the goals disclosed herein, the following
sequences were used:
TABLE-US-00008 SEQ. ID. NO.: 1 ##STR00001## ##STR00002##
##STR00003## ##STR00004## ##STR00005##
FYKSLRAEQTDAAVKNWMTQTLLIQNANPDCKLVLKGLGVNPTLEEMLTA
CQGVGGPGQKARLMAEALKEALAPVPIPFAAAQQRGPRKTIKCWNCGKEG
HSARQCRAPRRQGCWKCGKMDHVMAKCPDRQAGFLGLGPWGKKPRNFPMA
QVHQGLMPTAPPEDPAVDLLKNYMQLGKQQREKQRESRXKPYKEVTEDLL HLNSLFGGDQ.
(TO BE CONFIRMED, but in several seqs. of the GENBANK submitted by
David's group, the seqs. have 510 amino acids--the final Q residue
is missing).
[0058] SEQ. ID. NO.: 2 is a sequence of amino acids of the
recombinant protein Gag 45-269, cloned and expressed in the E/NS1
intergenic region by the yellow fever 17D virus. Gag45-269 fragment
is shaded in gray.
TABLE-US-00009 SEQ. ID. NO.: 2 ##STR00006## ##STR00007##
##STR00008## ##STR00009##
VFGSVYTTMFGGVSWMIRILIGFLVLWIGTNSRNTSMAMTCIAVGGITLF LGFTVGA
Wherein the other sequences present in SEQ. ID. NO.: 2 are
sequences of the yellow fever virus and correspond to:
TABLE-US-00010 SEQ. ID. NO.: 3 ##STR00010##
[0059] SEQ. ID. NO.: 3 is a variation of 10 amino acid residues of
the N-terminal sequence of NS1 protein of the yellow fever virus
(DQGAINFGK); YF genome position from 2453 to 2482)[sic] in which an
amino acid replacement was introduced (from cysteine to serine,
shaded in gray).
TABLE-US-00011 SEQ. ID. NO.: 4 KESSIG
[0060] SEQ. ID. NO.: 4 is the sequence of amino acids corresponding
both to the sequence of nucleotides of the position from 2145 to
2164 of the yellow fever virus genome and the motif comprising the
amino acid residues 391 to 397 of E protein of the yellow fever
virus.
TABLE-US-00012 SEQ. ID. NO.: .5
TSLGKAVHQVFGSVYTTMFGGVSWMIRILIGFLVLWIGTNSRNTSMAMTC
IAVGGITLFLGFTVGA
[0061] SEQ. ID. NO.: 5 is a variation of the last-terminal [sic]
amino acid residues of E protein of dengue virus serotype 4 (dengue
virus serotype 4 genome position from 2226 to 2423; GenBank
AF326825). SEQ. ID. NO.: 5 includes a replacement relative to the
original sequence where the amino acid terminal sequence VQA is
changed to VGA.
[0062] SEQ. ID. NO.: 6 is a nucleotide sequence (924 nucleotides)
of the cassette of recombinant Gag45-269, cloned in the E/NS1
intergenic region of the yellow fever 17D virus. Gag 45-269
fragment is shaded in gray.
TABLE-US-00013 SEQ. ID. NO.: 6 ##STR00011## ##STR00012##
##STR00013## ##STR00014## ##STR00015## ##STR00016## ##STR00017##
##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022##
ACCAGGTTTTTGGAAGTGTGTATACAACCATGTTTGGAGGAGTCTCATGG
ATGATTAGAATCCTAATTGGGTTCTTAGTGTTGTGGATTGGCACGAACTC
CAGGAACACTTCAATGGCTATGACGTGCATAGCTGTTGGAGGAATCACTC
TGTTTCTGGGCTTCACAGTTGGCGCC 3'
[0063] SEQ. ID. NO.: 7 is a sequence of 11, 785 [sic] nucleotides
of the recombinant virus genome taking cassette Gag 45-269, called
rYF17D/SIVGag45-269.
TABLE-US-00014 SEQ. ID. NO.: 7 5'
AGTAAATCCTGTGTGCTAATTGAGGTGCATTGGTCTGCAAATCGAGTTGC
TAGGCAATAAACACATTTGGATTAATTTTAATCGTTCGTTGAGCGATTAG
CAGAGAACTGACCAGAACATGTCTGGTCGTAAAGCTCAGGGAAAAACCCT
GGGCGTCAATATGGTACGACGAGGAGTTCGCTCCTTGTCAAACAAAATAA
AACAAAAAACAAAACAAATTGGAAACAGACCTGGACCTTCAAGAGGTGTT
CAAGGATTTATCTTTTTCTTTTTGTTCAACATTTTGACTGGAAAAAAGAT
CACAGCCCACCTAAAGAGGTTGTGGAAAATGCTGGACCCAAGACAAGGCT
TGGCTGTTCTAAGGAAAGTCAAGAGAGTGGTGGCCAGTTTGATGAGAGGA
TTGTCCTCAAGGAAACGCCGTTCCCATGATGTTCTGACTGTGCAATTCCT
AATTTTGGGAATGCTGTTGATGACCGGTGGAGTGACCTTGGTGCGGAAAA
ACAGATGGTTGCTCCTAAATGTGACATCTGAGGACCTCGGGAAAACATTC
TCTGTGGGCACAGGCAACTGCACAACAAACATTTTGGAAGCCAAGTACTG
GTGCCCAGACTCAATGGAATACAACTGTCCCAATCTCAGTCCAAGAGAGG
AGCCAGATGACATTGATTGCTGGTGCTATGGGGTGGAAAACGTTAGAGTC
GCATATGGTAAGTGTGACTCAGCAGGCAGGTCTAGGAGGTCAAGAAGGGC
CATTGACTTGCCTACGCATGAAAACCATGGTTTGAAGACCCGGCAAGAAA
AATGGATGACTGGAAGAATGGGTGAAAGGCAACTCCAAAAGATTGAGAGA
TGGTTCGTGAGGAACCCCTTTTTTGCAGTGACGGCTCTGACCATTGCCTA
CCTTGTGGGAAGCAACATGACGCAACGAGTCGTGATTGCCCTACTGGTCT
TGGCTGTTGGTCCGGCCTACTCAGCTCACTGCATTGGAATTACTGACAGG
GATTTCATTGAGGGGGTGCATGGAGGAACTTGGGTTTCAGCTACCCTGGA
GCAAGACAAGTGTGTCACTGTTATGGCCCCTGACAAGCCTTCATTGGACA
TCTCACTAGAGACAGTAGCCATTGATAGACCTGCTGAGGCGAGGAAAGTG
TGTTACAATGCAGTTCTCACTCATGTGAAGATTAATGACAAGTGCCCCAG
CACTGGAGAGGCCCACCTAGCTGAAGAGAACGAAGGGGACAATGCGTGCA
AGCGCACTTATTCTGATAGAGGCTGGGGCAATGGCTGTGGCCTATTTGGG
AAAGGGAGCATTGTGGCATGCGCCAAATTCACTTGTGCCAAATCCATGAG
TTTGTTTGAGGTTGATCAGACCAAAATTCAGTATGTCATCAGAGCACAAT
TGCATGTAGGGGCCAAGCAGGAAAATTGGAATACCAGCATTAAGACTCTC
AAGTTTGATGCCCTGTCAGGCTCCCAGGAAGTCGAGTTCATTGGGTATGG
AAAAGCTACACTGGAATGCCAGGTGCAAACTGCGGTGGACTTTGGTAACA
GTTACATCGCTGAGATGGAAACAGAGAGCTGGATAGTGGACAGACAGTGG
GCCCAGGACTTGACCCTGCCATGGCAGAGTGGAAGTGGCGGGGTGTGGAG
AGAGATGCATCATCTTGTCGAATTTGAACCTCCGCATGCCGCCACTATCA
GAGTACTGGCCCTGGGAAACCAGGAAGGCTCCTTGAAAACAGCTCTTACT
GGCGCAATGAGGGTTACAAAGGACACAAATGACAACAACCTTTACAAACT
ACATGGTGGACATGTTTCTTGCAGAGTGAAATTGTCAGCTTTGACACTCA
AGGGGACATCCTACAAAATATGCACTGACAAAATGTTTTTTGTCAAGAAC
CCAACTGACACTGGCCATGGCACTGTTGTGATGCAGGTGAAAGTGTCAAA
AGGAACCCCCTGCAGGATTCCAGTGATAGTAGCTGATGATCTTACAGCGG
CAATCAATAAAGGCATTTTGGTTACAGTTAACCCCATCGCCTCAACCAAT
GATGATGAAGTGCTGATTGAGGTGAACCCACCTTTTGGAGACAGCTACAT
TATCGTTGGGAGAGGAGATTCACGTCTCACTTACCAGTGGCACAAAGAGG
GAAGCTCAATAGGAAAGTTGTTCACTCAGACCATGAAAGGCGTGGAACGC
CTGGCCGTCATGGGAGACACCGCCTGGGATTTCAGCTCCGCTGGAGGGTT
CTTCACTTCGGTTGGGAAAGGAATTCATACGGTGTTTGGCTCTGCCTTTC
AGGGGCTATTTGGCGGCTTGAACTGGATAACAAAGGTCATCATGGGGGCG
GTACTCATATGGGTTGGCATCAACACAAGAAACATGACAATGTCCATGAG
CATGATCTTGGTAGGAGTGATCATGATGTTTTTGTCTCTAGGAGTTGGGG ##STR00023##
##STR00024## ##STR00025## ##STR00026## ##STR00027## ##STR00028##
##STR00029## ##STR00030## ##STR00031## ##STR00032## ##STR00033##
##STR00034## ##STR00035## ##STR00036## ##STR00037## ##STR00038##
##STR00039## GGCAAGAGAGAGCTCAAGTGCGGAGATGGTATCTTCATATTTAGAGACTCT
GATGACTGGCTGAACAAGTACTCATACTATCCAGAAGATCCTGTGAAGCTT
GCATCAATAGTGAAAGCCTCTTTCGAAGAAGGGAAGTGTGGCCTAAATTCA
GTTGACTCCCTTGAGCATGAGATGTGGAGAAGCAGGGCAGATGAGATTAAT
ACCATTTTTGAGGAAAACGAGGTGGACATTTCTGTTGTCGTGCAGGATCCA
AAGAATGTTTACCAGAGAGGAACTCATCCATTTTCCAGAATTCGGGATGGT
CTGCAGTATGGTTGGAAGACTTGGGGTAAGAACCTTGTGTTCTCCCCAGGG
AGGAAGAATGGAAGCTTCATCATAGATGGAAAGTCCAGGAAAGAATGCCCG
TTTTCAAACCGGGTCTGGAATTCTTTCCAGATAGAGGAGTTTGGGACGGGA
GTGTTCACCACACGCGTGTACATGGACGCAGTCTTTGAATACACCATAGAC
TGCGATGGATCTATCTTGGGTGCAGCGGTGAACGGAAAAAAGAGTGCCCAT
GGCTCTCCAACATTTTGGATGGGAAGTCATGAAGTAAATGGGACATGGATG
ATCCACACCTTGGAGGCATTAGATTACAAGGAGTGTGAGTGGCCACTGACA
CATACGATTGGAACATCAGTTGAAGAGAGTGAAATGTTCATGCCGAGATCA
ATCGGAGGCCCAGTTAGCTCTCACAATCATATCCCTGGATACAAGGTTCAG
ACGAACGGACCTTGGATGCAGGTACCACTAGAAGTGAAGAGAGAAGCTTGC
CCAGGGACTAGCGTGATCATTGATGGCAACTGTGATGGACGGGGAAAATCA
ACCAGATCCACCACGGATAGCGGGAAAGTTATTCCTGAATGGTGTTGCCGC
TCCTGCACAATGCCGCCTGTGAGCTTCCATGGTAGTGATGGGTGTTGGTAT
CCCATGGAAATTAGGCCAAGGAAAACGCATGAAAGCCATCTGGTGCGCTCC
TGGGTTACAGCTGGAGAAATACATGCTGTCCCTTTTGGTTTGGTGAGCATG
ATGATAGCAATGGAAGTGGTCCTAAGGAAAAGACAGGGACCAAAGCAAATG
TTGGTTGGAGGAGTAGTGCTCTTGGGAGCAATGCTGGTCGGGCAAGTAACT
CTCCTTGATTTGCTGAAACTCACAGTGGCTGTGGGATTGCATTTCCATGAG
ATGAACAATGGAGGAGACGCCATGTATATGGCGTTGATTGCTGCCTTTTCA
ATCAGACCAGGGCTGCTCATCGGCTTTGGGCTCAGGACCCTATGGAGCCCT
CGGGAACGCCTTGTGCTGACCCTAGGAGCAGCCATGGTGGAGATTGCCTTG
GGTGGCGTGATGGGCGGCCTGTGGAAGTATCTAAATGCAGTTTCTCTCTGC
ATCCTGACAATAAATGCTGTTGCTTCTAGGAAAGCATCAAATACCATCTTG
CCCCTCATGGCTCTGTTGACACCTGTCACTATGGCTGAGGTGAGACTTGCC
GCAATGTTCTTTTGTGCCATGGTTATCATAGGGGTCCTTCACCAGAATTTC
AAGGACACCTCCATGCAGAAGACTATACCTCTGGTGGCCCTCACACTCACA
TCTTACCTGGGCTTGACACAACCTTTTTTGGGCCTGTGTGCATTTCTGGCA
ACCCGCATATTTGGGCGAAGGAGTATCCCAGTGAATGAGGCACTCGCAGCA
GCTGGTCTAGTGGGAGTGCTGGCAGGACTGGCTTTTCAGGAGATGGAGAAC
TTCCTTGGTCCGATTGCAGTTGGAGGACTCCTGATGATGCTGGTTAGCGTG
GCTGGGAGGGTGGATGGGCTAGAGCTCAAGAAGCTTGGTGAAGTTTCATGG
GAAGAGGAGGCGGAGATCAGCGGGAGTTCCGCCCGCTATGATGTGGCACTC
AGTGAACAAGGGGAGTTCAAGCTGCTTTCTGAAGAGAAAGTGCCATGGGAC
CAGGTTGTGATGACCTCGCTGGCCTTGGTTGGGGCTGCCCTCCATCCATTT
GCTCTTCTGCTGGTCCTTGCTGGGTGGCTGTTTCATGTCAGGGGAGCTAGG
AGAAGTGGGGATGTCTTGTGGGATATTCCCACTCCTAAGATCATCGAGGAA
TGTGAACATCTGGAGGATGGGATTTATGGCATATTCCAGTCAACCTTCTTG
GGGGCCTCCCAGCGAGGAGTGGGAGTGGCACAGGGAGGGGTGTTCCACACA
ATGTGGCATGTCACAAGAGGAGCTTTCCTTGTCAGGAATGGCAAGAAGTTG
ATTCCATCTTGGGCTTCAGTAAAGGAAGACCTTGTCGCCTATGGTGGCTCA
TGGAAGTTGGAAGGCAGATGGGATGGAGAGGAAGAGGTCCAGTTGATCGCG
GCTGTTCCAGGAAAGAACGTGGTCAACGTCCAGACAAAACCGAGCTTGTTC
AAAGTGAGGAATGGGGGAGAAATCGGGGCTGTCGCTCTTGACTATCCGAGT
GGCACTTCAGGATCTCCTATTGTTAACAGGAACGGAGAGGTGATTGGGCTG
TACGGCAATGGCATCCTTGTCGGTGACAACTCCTTCGTGTCCGCCATATCC
CAGACTGAGGTGAAGGAAGAAGGAAAGGAGGAGCTCCAAGAGATCCCGACA
ATGCTAAAGAAAGGAATGACAACTGTCCTTGATTTTCATCCTGGAGCTGGG
AAGACAAGACGTTTCCTCCCACAGATCTTGGCCGAGTGCGCACGGAGACGC
TTGCGCACTCTTGTGTTGGCCCCCACCAGGGTTGTTCTTTCTGAAATGAAG
GAGGCTTTTCACGGCCTGGACGTGAAATTCCACACACAGGCTTTTTCCGCT
CACGGCAGCGGGAGAGAAGTCATTGATGCCATGTGCCATGCCACCCTAACT
TACAGGATGTTGGAACCAACTAGGGTTGTTAACTGGGAAGTGATCATTATG
GATGAAGCCCATTTTTTGGATCCAGCTAGCATAGCCGCTAGAGGTTGGGCA
GCGCACAGAGCTAGGGCAAATGAAAGTGCAACAATCTTGATGACAGCCACA
CCGCCTGGGACTAGTGATGAATTTCCACATTCAAATGGTGAAATAGAAGAT
GTTCAAACGGACATACCCAGTGAGCCCTGGAACACAGGGCATGACTGGATC
CTGGCTGACAAAAGGCCCACGGCATGGTTCCTTCCATCCATCAGAGCTGCA
AATGTCATGGCTGCCTCTTTGCGTAAGGCTGGAAAGAGTGTGGTGGTCCTG
AACAGGAAAACCTTTGAGAGAGAATACCCCACGATAAAGCAGAAGAAACCT
GACTTTATATTGGCCACTGACATAGCTGAAATGGGAGCCAACCTTTGCGTG
GAGCGAGTGCTGGATTGCAGGACGGCTTTTAAGCCTGTGCTTGTGGATGAA
GGGAGGAAGGTGGCAATAAAAGGGCCACTTCGTATCTCCGCATCCTCTGCT
GCTCAAAGGAGGGGGCGCATTGGGAGAAATCCCAACAGAGATGGAGACTCA
TACTACTATTCTGAGCCTACAAGTGAAAATAATGCCCACCACGTCTGCTGG
TTGGAGGCCTCAATGCTCTTGGACAACATGGAGGTGAGGGGTGGAATGGTC
GCCCCACTCTATGGCGTTGAAGGAACTAAAACACCAGTTTCCCCTGGTGAA
ATGAGACTGAGGGATGACCAGAGGAAAGTCTTCAGAGAACTAGTGAGGAAT
TGTGACCTGCCCGTTTGGCTTTCGTGGCAAGTGGCCAAGGCTGGTTTGAAG
ACGAATGATCGTAAGTGGTGTTTTGAAGGCCCTGAGGAACATGAGATCTTG
AATGACAGCGGTGAAACAGTGAAGTGCAGGGCTCCTGGAGGAGCAAAGAAG
CCTCTGCGCCCAAGGTGGTGTGATGAAAGGGTGTCATCTGACCAGAGTGCG
CTGTCTGAATTTATTAAGTTTGCTGAAGGTAGGAGGGGAGCTGCTGAAGTG
CTAGTTGTGCTGAGTGAACTCCCTGATTTCCTGGCTAAAAAAGGTGGAGAG
GCAATGGATACCATCAGTGTGTTCCTCCACTCTGAGGAAGGCTCTAGGGCT
TACCGCAATGCACTATCAATGATGCCTGAGGCAATGACAATAGTCATGCTG
TTTATACTGGCTGGACTACTGACATCGGGAATGGTCATCTTTTTCATGTCT
CCCAAAGGCATCAGTAGAATGTCTATGGCGATGGGCACAATGGCCGGCTGT
GGATATCTCATGTTCCTTGGAGGCGTCAAACCCACTCACATCTCCTATGTC
ATGCTCATATTCTTTGTCCTGATGGTGGTTGTGATCCCCGAGCCAGGGCAA
CAAAGGTCCATCCAAGACAACCAAGTGGCATACCTCATTATTGGCATCCTG
ACGCTGGTTTCAGCGGTGGCAGCCAACGAGCTAGGCATGCTGGAGAAAACC
AAAGAGGACCTCTTTGGGAAGAAGAACTTAATTCCATCTAGTGCTTCACCC
TGGAGTTGGCCGGATCTTGACCTGAAGCCAGGAGCTGCCTGGACAGTGTAC
GTTGGCATTGTTACAATGCTCTCTCCAATGTTGCACCACTGGATCAAAGTC
GAATATGGCAACCTGTCTCTGTCTGGAATAGCCCAGTCAGCCTCAGTCCTT
TCTTTCATGGACAAGGGGATACCATTCATGAAGATGAATATCTCGGTCATA
ATGCTGCTGGTCAGTGGCTGGAATTCAATAACAGTGATGCCTCTGCTCTGT
GGCATAGGGTGCGCCATGCTCCACTGGTCTCTCATTTTACCTGGAATCAAA
GCGCAGCAGTCAAAGCTTGCACAGAGAAGGGTGTTCCATGGCGTTGCCAAG
AACCCTGTGGTTGATGGGAATCCAACAGTTGACATTGAGGAAGCTCCTGAA
ATGCCTGCCCTTTATGAGAAGAAACTGGCTCTATATCTCCTTCTTGCTCTC
AGCCTAGCTTCTGTTGCCATGTGCAGAACGCCCTTTTCATTGGCTGAAGGC
ATTGTCCTAGCATCAGCTGCCTTAGGGCCGCTCATAGAGGGAAACACCAGC
CTTCTTTGGAATGGACCCATGGCTGTCTCCATGACAGGAGTCATGAGGGGG
AATCACTATGCTTTTGTGGGAGTCATGTACAATCTATGGAAGATGAAAACT
GGACGCCGGGGGAGCGCGAATGGAAAAACTTTGGGTGAAGTCTGGAAGAGG
GAACTGAATCTGTTGGACAAGCGACAGTTTGAGTTGTATAAAAGGACCGAC
ATTGTGGAGGTGGATCGTGATACGGCACGCAGGCATTTGGCCGAAGGGAAG
GTGGACACCGGGGTGGCGGTCTCCAGGGGGACCGCAAAGTTAAGGTGGTTC
CATGAGCGTGGCTATGTCAAGCTGGAAGGTAGGGTGATTGACCTGGGGTGT
GGCCGCGGAGGCTGGTGTTACTACGCTGCTGCGCAAAAGGAAGTGAGTGGG
GTCAAAGGATTTACTCTTGGAAGAGACGGCCATGAGAAACCCATGAATGTG
CAAAGTCTGGGATGGAACATCATCACCTTCAAGGACAAAACTGATATCCAC
CGCCTAGAACCAGTGAAATGTGACACCCTTTTGTGTGACATTGGAGAGTCA
TCATCGTCATCGGTCACAGAGGGGGAAAGGACCGTGAGAGTTCTTGATACT
GTAGAAAAATGGCTGGCTTGTGGGGTTGACAACTTCTGTGTGAAGGTGTTA
GCTCCATACATGCCAGATGTTCTTGAGAAACTGGAATTGCTCCAAAGGAGG
TTTGGCGGAACAGTGATCAGGAACCCTCTCTCCAGGAATTCCACTCATGAA
ATGTACTACGTGTCTGGAGCCCGCAGCAATGTCACATTTACTGTGAACCAA
ACATCCCGCCTCCTGATGAGGAGAATGAGGCGTCCAACTGGAAAAGTGACC
CTGGAGGCTGACGTCATCCTCCCAATTGGGACACGCAGTGTTGAGACAGAC
AAGGGACCCCTGGACAAAGAGGCCATAGAAGAAAGGGTTGAGAGGATAAAA
TCTGAGTACATGACCTCTTGGTTTTATGACAATGACAACCCCTACAGGACC
TGGCACTACTGTGGCTCCTATGTCACAAAAACCTCAGGAAGTGCGGCGAGC
ATGGTAAATGGTGTTATTAAAATTCTGACATATCCATGGGACAGGATAGAG
GAGGTCACCAGAATGGCAATGACTGACACAACCCCTTTTGGACAGCAAAGA
GTGTTTAAAGAAAAAGTTGACACCAGAGCAAAGGATCCACCAGCGGGAACT
AGGAAGATCATGAAAGTTGTCAACAGGTGGCTGTTCCGCCACCTGGCCAGA
GAAAAGAGCCCCAGACTGTGCACAAAGGAAGAATTTATTGCAAAAGTCCGA
AGTCATGCAGCCATTGGAGCTTACCTGGAAGAACAAGAACAGTGGAAGACT
GCCAATGAGGCTGTCCAAGACCCAAAGTTCTGGGAACTGGTGGATGAAGAA
AGGAAGCTGCACCAACAAGGCAGGTGTCGGACTTGTGTGTACAACATGATG
GGGAAAAGAGAGAAGAAGCTGTCAGAGTTTGGGAAAGCAAAGGGAAGCCGT
GCCATATGGTATATGTGGCTGGGAGCGCGGTATCTTGAGTTTGAGGCCCTG
GGATTCCTGAATGAGGACCATTGGGCTTCCAGGGAAAACTCAGGAGGAGGA
GTGGAAGGCATTGGCTTACAATACCTAGGATATGTGATCAGAGACCTGGCT
GCAATGGATGGTGGTGGATTCTACGCGGATGACACCGCTGGATGGGACACG
CGCATCACAGAGGCAGACCTTGATGATGAACAGGAGATCTTGAACTACATG
AGCCCACATCACAAAAAACTGGCACAAGCAGTGATGGAAATGACATACAAG
AACAAAGTGGTGAAAGTGTTGAGACCAGCCCCAGGAGGGAAAGCCTACATG
GATGTCATAAGTCGACGAGACCAGAGAGGATCCGGGCAGGTAGTGACTTAT
GCTCTGAACACCATCACCAACTTGAAAGTCCAATTGATCAGAATGGCAGAA
GCAGAGATGGTGATACATCACCAACATGTTCAAGATTGTGATGAATCAGTT
CTGACCAGGCTGGAGGCATGGCTCACTGAGCACGGATGTAACAGACTGAAG
AGGATGGCGGTGAGTGGAGACGACTGTGTGGTCCGGCCCATCGATGACAGG
TTCGGCCTGGCCCTGTCCCATCTCAACGCCATGTCCAAGGTTAGAAAGGAC
ATATCTGAATGGCAGCCATCAAAAGGGTGGAATGATTGGGAGAATGTGCCC
TTCTGTTCCCACCACTTCCATGAACTACAGCTGAAGGATGGCAGGAGGATT
GTGGTGCCTTGCCGAGAACAGGACGAGCTCATTGGGAGAGGAAGGGTGTCT
CCAGGAAACGGCTGGATGATCAAGGAAACAGCTTGCCTCAGCAAAGCCTAT
GCCAACATGTGGTCACTGATGTATTTTCACAAAAGGGACATGAGGCTACTG
TCATTGGCTGTTTCCTCAGCTGTTCCCACCTCATGGGTTCCACAAGGACGC
ACAACATGGTCGATTCATGGGAAAGGGGAGTGGATGACCACGGAAGACATG
CTTGAGGTGTGGAACAGAGTATGGATAACCAACAACCCACACATGCAGGAC
AAGACAATGGTGAAAAAATGGAGAGATGTCCCTTATCTAACCAAGAGACAA
GACAAGCTGTGCGGATCACTGATTGGAATGACCAATAGGGCCACCTGGGCC
TCCCACATCCATTTAGTCATCCATCGTATCCGAACGCTGATTGGACAGGAG
AAATACACTGACTACCTAACAGTCATGGACAGGTATTCTGTGGATGCTGAC
CTGCAACTGGGTGAGCTTATCTGAAACACCATCTAACAGGAATAACCGGGA
TACAAACCACGGGTGGAGAACCGGACTCCCCACAACCTGAAACCGGGATAT
AAACCACGGCTGGAGAACCGGGCTCCGCACTTAAAATGAAACAGAAACCGG
GATAAAAACTACGGATGGAGAACCGGACTCCACACATTGAGACAGAAGAAG
TTGTCAGCCCAGAACCCCACACGAGTTTTGCCACTGCTAAGCTGTGAGGCA
GTGCAGGCTGGGACAGCCGACCTCCAGGTTGCGAAAAACCTGGTTTCTGGG
ACCTCCCACCCCAGAGTAAAAAGAACGGAGCCTCCGCTACCACCCTCCCAC
GTGGTGGTAGAAAGACGGGGTCTAGAGGTTAGAGGAGACCCTCCAGGGAAC
AAATAGTGGGACCATATTGACGCCAGGGAAAGACCGGAGTGGTTCTCTGCT
TTTCCTCCAGAGGTCTGTGAGCACAGTTTGCTCAAGAATAAGCAGACCTTT
GGATGACAAACACAAAACCAC 3'
[0064] SEQ. ID. NO.: 8 is a precursory sequence of the polyprotein
of virus rYF17D/SIVGag45-269. The recombinant protein is shaded in
gray.
TABLE-US-00015 SEQ. ID. NO.: 8
SGRKAQGKTLGVNMVRRGVRSLSNKIKQKTKQIGNRPGPSRGVQGFIFFF
LFNILTGKKITAHLKRLWKMLDPRQGLAVLRKVKRVVASLMRGLSSRKRR
SHDVLTVQFLILGMLLMTGGVTLVRKNRWLLLNVTSEDLGKTFSVGTGNC
TTNILEAKYWCPDSMEYNCPNLSPREEPDDIDCWCYGVENVRVAYGKCDS
AGRSRRSRRAIDLPTHENHGLKTRQEKWMTGRMGERQLQKIERWFVRNPF
FAVTALTIAYLVGSNMTQRVVIALLVLAVGPAYSAHCIGITDRDFIEGVH
GGTWVSATLEQDKCVTVMAPDKPSLDISLETVAIDRPAEARKVCYNAVLT
HVKINDKCPSTGEAHLAEENEGDNACKRTYSDRGWGNGCGLFGKGSIVAC
AKFTCAKSMSLFEVDQTKIQYVIRAQLHVGAKQENWNTSIKTLKFDALSG
SQEVEFIGYGKATLECQVQTAVDFGNSYIAEMETESWIVDRQWAQDLTLP
WQSGSGGVWREMHHLVEFEPPHAATIRVLALGNQEGSLKTALTGAMRVTK
DTNDNNLYKLHGGHVSCRVKLSALTLKGTSYKICTDKMFFVKNPTDTGHG
TVVMQVKVSKGTPCRIPVIVADDLTAAINKGILVTVNPIASTNDDEVLIE
VNPPFGDSYTIVGRGDSRLTYQWHKEGSSIGKLFTQTMKGVERLAVMGDT
AWDFSSAGGFFTSVGKGIHTVFGSAFQGLFGGLNWITKVIMGAVLIWVGI ##STR00040##
##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045##
GDGIFIFRDSDDWLNKYSYYPEDPVKLASIVKASFEEGKCGLNSVDSLEH
EMWRSRADEINTIFEENEVDISVVVQDPKNVYQRGTHPFSRIRDGLQYGW
KTWGKNLVFSPGRKNGSFIIDGKSRKECPFSNRVWNSFQIEEFGTGVETT
RVYMDAVFEYTIDCDGSILGAAVNGKKSAHGSPTFWMGSHEVNGTWMIHT
LEALDYKECEWPLTHTIGTSVEESEMFMPRSIGGPVSSHNHIPGYKVQTN
GPWMQVPLEVKREACPGTSVIIDGNCDGRGKSTRSTTDSGKVIPEWCCRS
CTMPPVSFHGSDGCWYPMEIRPRKTHESHLVRSWVTAGEIHAVPFGLVSM
MIAMEVVLRKRQGPKQMLVGGVVLLGAMLVGQVTLLDLLKLTVAVGLHFH
EMNNGGDAMYMALIAAFSIRPGLLIGFGLRTLWSPRERLVLTLGAAMVEI
ALGGVMGGLWKYLNAVSLCILTINAVASRKASNTILPLMALLTPVTMAEV
RLAAMFFCAMVIIGVLHQNFKDTSMQKTIPLVALTLTSYLGLTQPFLGLC
AFLATRIFGRRSIPVNEALAAAGLVGVLAGLAFQEMENFLGPIAVGGLLM
MLVSVAGRVDGLELKKLGEVSWEEEAEISGSSARYDVALSEQGEFKLLSE
EKVPWDQVVMTSLALVGAALHPFALLLVLAGWLFHVRGARRSGDVLWDIP
TPKIIEECEHLEDGIYGIFQSTFLGASQRGVGVAQGGVFHTMWHVTRGAF
LVRNGKKLIPSWASVKEDLVAYGGSWKLEGRWDGEEEVQLIAAVPGKNVV
NVQTKPSLFKVRNGGEIGAVALDYPSGTSGSPIVNRNGEVIGLYGNGILV
GDNSFVSAISQTEVKEEGKEELQEIPTMLKKGMTTVLDFHPGAGKTRRFL
PQILAECARRRLRTLVLAPTRVVLSEMKEAFHGLDVKFHTQAFSAHGSGR
EVIDAMCHATLTYRMLEPTRVVNWEVIIMDEAHFLDPASIAARGWAAHRA
RANESATILMTATPPGTSDEFPHSNGEIEDVQTDIPSEPWNTGHDWILAD
KRPTAWFLPSIRAANVMAASLRKAGKSVVVLNRKTFEREYPTIKQKKPDF
ILATDIAEMGANLCVERVLDCRTAFKPVLVDEGRKVAIKGPLRISASSAA
QRRGRIGRNPNRDGDSYYYSEPTSENNAHHVCWLEASMLLDNMEVRGGMV
APLYGVEGTKTPVSPGEMRLRDDQRKVFRELVRNCDLPVWLSWQVAKAGL
KTNDRKWCFEGPEEHEILNDSGETVKCRAPGGAKKPLRPRWCDERVSSDQ
SALSEFIKFAEGRRGAAEVLVVLSELPDFLAKKGGEAMDTISVFLHSEEG
SRAYRNALSMMPEAMTIVMLFILAGLLTSGMVIFFMSPKGISRMSMAMGT
MAGCGYLMFLGGVKPTHISYVMLIFFVLMVVVIPEPGQQRSIQDNQVAYL
IIGILTLVSAVAANELGMLEKTKEDLFGKKNLIPSSASPWSWPDLDLKPG
AAWTVYVGIVTMLSPMLHHWIKVEYGNLSLSGIAQSASVLSFMDKGIPFM
KMNISVIMLLVSGWNSITVMPLLCGIGCAMLHWSLILPGIKAQQSKLAQR
RVFHGVAKNPVVDGNPTVDIEEAPEMPALYEKKLALYLLLALSLASVAMC
RTPFSLAEGIVLASAALGPLIEGNTSLLWNGPMAVSMTGVMRGNHYAFVG
VMYNLWKMKTGRRGSANGKTLGEVWKRELNLLDKRQFELYKRTDIVEVDR
DTARRHLAEGKVDTGVAVSRGTAKLRWFHERGYVKLEGRVIDLGCGRGGW
CYYAAAQKEVSGVKGFTLGRDGHEKPMNVQSLGWNIITFKDKTDIHRLEP
VKCDTLLCDIGESSSSSVTEGERTVRVLDTVEKWLACGVDNFCVKVLAPY
MPDVLEKLELLQRRFGGTVIRNPLSRNSTHEMYYVSGARSNVTFTVNQTS
RLLMRRMRRPTGKVTLEADVILPIGTRSVETDKGPLDKEAIEERVERIKS
EYMTSWFYDNDNPYRTWHYCGSYVTKTSGSAASMVNGVIKILTYPWDRIE
EVTRMAMTDTTPFGQQRVFKEKVDTRAKDPPAGTRKIMKVVNRWLFRHLA
REKSPRLCTKEEFIAKVRSHAATGAYLEEQEQWKTANEAVQDPKFWELVD
EERKLHQQGRCRTCVYNMMGKREKKLSEFGKAKGSRAIWYMWLGARYLEF
EALGFLNEDHWASRENSGGGVEGIGLQYLGYVIRDLAAMDGGGFYADDTA
GWDTRITEADLDDEQEILNYMSPHHKKLAQAVMEMTYKNKVVKVLRPAPG
GKAYMDVISRRDQRGSGQVVTYALNTITNLKVQLIRMAEAEMVIHHQHVQ
DCDESVLTRLEAWLTEHGCNRLKRMAVSGDDCVVRPIDDRFGLALSHLNA
MSKVRKDISEWQPSKGWNDWENVPFCSHHFHELQLKDGRRIVVPCREQDE
LIGRGRVSPGNGWMIKETACLSKAYANMWSLMYFHKRDMRLLSLAVSSAV
PTSWVPQGRTTWSIHGKGEWMTTEDMLEVWNRVWITNNPHMQDKTMVKKW
RDVPYLTKRQDKLCGSLIGMTNRATWASHIHLVIHRIRTLIGQEKYTDYL
TVMDRYSVDADLQLGEL
[0065] Other appropriate sequences to prepare the recombinant
yellow fever viruses contemplated herein include corresponding
sequences in HIV (for example, complete Gag polypeptide or a
fragment thereof). For example, the heterologous polypeptide
expressed by the recombinant yellow fever viruses considered here
may comprise a fragment of SEQ. ID. NO.: 9, or a related protein
(for example, a protein having at least about 50%, 60%, 70%, 80%,
90%, 95%, 96%, 97%, 98%, or 99% of amino acid sequence identity
with SEQ. ID. NO.: 9).
TABLE-US-00016 SED. [sic] ID. NO.: 9 MGARASVLSG GKLDKWEKIR
LRPGGKKTYQ LKHIVWASRE LERFAVNPGL LETGGGCKQI LVQLQPSLQT GSEELKSLYN
AVATLYCVHQ GIEVRDTKEA LDKIEEEQNK SKKKAQQAAA DTGNSSQVSQ NYPIVQNLQG
QMVHQAISPR TLNAWVKVIE EKAFSPEVIP MFSALSEGAT PQDLNTMLNT VGGHQAAMQM
LKETINEEAA EWDRLHPAHA GPNAPGQMRE PRGSDIAGTT STLQEQIGWM TSNPPVPVGE
IYKRWIILGL NKIVRMYSPV SILDIRQGPK EPFRDYVDRF YKTLRAEQAS QDVKNWMTET
LLVQNANPDC KTILKALGPA ATLEEMMTAC QGVGGPSHKA RILAEAMSQV TSPANIMMQR
GNFRNQRKTI KCFNCGKEGH LARHCRAPRK KGCWKCGREG HQMKDCTERQ ANFLGKIWPS
HKGRPGNFLQ SRPEPTAPPE ESFRFGEETT TPPQKQEPLP SQKQETIDKD LYPLASLKSL
FGNDPSLQ formatting
[0066] A fragment, as considered here, may include at least 8, 9,
10, 20, 30, 40, 50 [sic] 100, or 200 contiguous amino acids of SEQ.
ID. NO.: 9. In one embodiment, the fragment comprises amino acids
45-269 of SEQ. ID. NO.: 9. The heterologous polypeptide may still
comprise a terminal amino acid sequence comprising KESSIG.
EXAMPLES
[0067] The present invention will now be described in detail by the
examples presented below. It is worth emphasizing that the
invention is not limited to these examples, and also includes
variations and modifications within the limits allowing its
action.
Example 1
Proliferation Properties of Recombinant rYF17D/SIVGag45-269 In Vero
Cells
[0068] The growth capacity of recombinant virus rYF17D/SIVGag45-269
was assessed and compared with other two viruses, YF 17DD and
YF17D/G1/2 T3 vaccine (the latter corresponding to the parental
vector virus, that is, heterologous insertion). Three independent
experiments of virus growth in monolayers of Vero cells were
performed. All experiments were performed in low MOI of 0.02 with
densities of Vero cells of 62,500 cells per cm.sup.2.
[0069] The recombinant virus rYF17D/SIVGag45-269 presented a lower
growth rate compared with the viruses of 17D vaccine, and had its
peak at 72 hours after the infection, exhibiting a titer of
6.61.+-.0.23 log 10 PFU/mL. FIG. 1 shows the viral growth curves in
Vero cells. Cells were infected with YF 17D control (gray
lozenges), YF17D/G1/2T3 virus (black squares), or with the
recombinant virus rYF17D/SIVGag45-269 (gray triangles in MOI of
0.02). Each time point represents the mean titer obtained for these
three experiments separately, with the respective standard
deviations.
Table 8 below shows the viral growth curves in Vero cells. Viral
titers (log 10 PFU/mL) are shown in each time point. Viral growth
peaks are shown in bold.
TABLE-US-00017 TABLE 8 Virus 24 h 48 h 72 h 96 h 120 h 144 h
YF17D/17DD 3.60 .+-. 0.54 6.14 .+-. 0.49 7.62 .+-. 1.02 7.63 .+-.
1.03 6.97 .+-. 0.62 7.30 .+-. 0.59 YF/7D/G1/2T3 5.32 .+-. 0.53 7.60
.+-. 1.03 7.45 .+-. 0.33 7.19 .+-. 0.27 6.95 .+-. 0.27 7.23 .+-.
0.40 rYF 17D SIV Gag 3.74 .+-. 0.35 5.57 .+-. 0.51 6.61 .+-. 0.23
6.30 .+-. 0.45 6.04 .+-. 0.27 5.61 .+-. 0.64 45-269
Example 2
Preliminary Immunological Studies of the YF17D Vaccine Virus
Infection in Mamu-A*01-Positive Rhesus Monkeys
[0070] First, the YF17D vaccine virus was used to infect four
Mamu-A*01-positive monkeys. The vaccine virus replicated in these
four animals and induced neutralizing antibodies in all four
monkeys by two weeks after vaccination (FIGS. 2A and 2B). To
monitor the CD8+ T-cell immune response against YF17D, its proteome
was scanned for peptides that might bind to Mamu-A*01 using
algorithms (MHC Pathway) [14]. The 52 YF17D-derived peptides which
were most likely to bind to Mamu-A*01, based on their predicted
affinity for this MHC class I molecule, were synthesized. IFNy
ELISPOT assay was used to screen these peptides in YF17D-infected
animals, and four Mamu-A*01-binding peptides: 17D 7844 (LTPVTMAEV;
LV91285-1293), 17D 7846 (VSPGNGWMI; V193250-3258), 17D 7850
(MSPKGISRM; MM92179-2187), and 17D 7858 (TTPFGQQRVF; TF102853-2863)
were recognized in vivo (FIG. 2C). Using a previously reported
protocol [15], the activation of CD8+ T cells was also observed in
all four animals (FIGS. 2D and 2E). Thus, as previously observed,
the YF17D vaccine virus replicates in Indian rhesus monkeys [16]
and induces neutralizing antibodies, yellow fever-specific
Mamu-A*01-restricted CD8+ T-cell responses, and CD8+ T-cell
activation. FIG. 2 shows that YF17D replicates and induces
neutralizing antibodies, virus-specific CD8+ T cells, and CD8+
T-cell activation in rhesus monkeys. FIG. 2A shows the replication
of YF17D during the first 10 days after vaccination with two
different doses, as measured by Q-PCR using YF17D-specific primers.
FIG. 2B shows the titer of neutralizing antibodies determined at 2
and 5 weeks after YF17D vaccination. In FIG. 2C, the fresh PBMC
from vaccinees (100,000 cells/well) were used in IFN-y ELISPOT
assays [17] to assess T-cell responses against YF17D. Four epitopes
(17D 7844, 17D 7846, 17D 7850, and 17D 7858)--predicted to bind to
Mamu-A*01 as defined by the algorithm (MHC Pathway)--were selected
for further studies. FIG. 2D presents the identification of
activated CD8+ T cells after vaccination with YF17D based on the
expression of proliferation and proapoptotic markers Ki-67 and
Bcl-2 [15]. Whole blood cells were stained with antibodies against
CD3 and CD8, and then permeabilized, and subsequently labeled with
Bcl-2- and Ki-67-specific antibodies. The flow cytometry graphs
were gated on CD3+ and CD8+ lymphocytes. FIG. E reveals the
expression kinetics of Ki-67 and Bcl-2 in T cells after vaccination
with YF17D.
Example 3
Immunogenicity of rYF17D/SIVGag45-269 in Rhesus Monkeys
[0071] In the following step, the YF17D vaccine virus was
engineered to express amino acids 45-269 of SIVmac239Gag
(rYF17D/SIVGag45-269) by inserting a yellow fever codon-optimized
sequence between the genes encoding the viral proteins E and NS1.
This recombinant virus replicated and induced neutralizing
antibodies in mice (data not shown). The rYF17D/SIVGag45-269
construct was then tested in six Mamu-A*01-positive Indian rhesus
monkeys. Evidence for the viral replication of rYF17D/SIVGag45-269
was found for five of these six monkeys (FIG. 3A). Neutralizing
antibodies were evident at two weeks after vaccination (FIG. 3B.
[sic] A single immunization with rYF17D/SIVGag45-269 induced
antigen-specific CD8+ T cells in five out of the six
Mamu-A*01-positive monkeys (FIG. 3C). This recombinant virus also
induced CD8+ T-cell activation in the majority of the vaccinated
animals (FIG. 3D). The sixth monkey (r04091) required a booster
dose 28 days after the first immunization to induce specific CD8+ T
cells (data not shown and FIG. 3C). No differences were found in
vaccine-induced immune responses between the animals vaccinated
with YF17D and those vaccinated with rYF17D/SIVGag45-269. There
was, however, considerable animal-to-animal variability. Animal
r02034, vaccinated with YF17D, exhibited massive CD8+ T-cell
activation (a peak of 35% at day 12; FIG. 2E), which was probably
induced by the high levels of viral replication (16,800 copies/mL
at day 5; FIG. 2A). The comparison of neutralizing antibody
responses at 2 and 5 weeks after vaccination in animals vaccinated
with YF17D or rYF17D/SIVGag45-269 did not reveal detectable
differences regarding the capacity of these two vaccines to induce
neutralizing antibodies (FIGS. 2B and 3B). It was also difficult to
detect differences in YF17D-specific CD8+ T-cell responses induced
by these two vaccines. Peak Mamu-A*01-restricted CD8+ T-cell
responses against YF17D ranged from barely detectable (r02110 at
day 24; FIG. 2C) to 400 SFCs/10.sup.6 PBMC (r04113 at day 12; FIG.
2C). Two of the animals that were vaccinated with
rYF17D/SIVGag45-269 did not originate CD8+ T-cell responses against
the Mamu-A*01-bound YF17D peptides, whereas this kind of response
was observed in four animals (FIG. 3C), with SFCs/10.sup.6 PBMC
ranging from 50 to 200. For almost every animal vaccinated with
rYF17D/SIVGag45-269, the Gag CM9-specific responses (peak in
50-750) were higher than those generated against the
Mamu-A*01-restricted YF17D epitopes (peak in 0-200), suggesting
that the recombinant virus replicated in a stable manner in vivo.
Thus, the recombinant YF17D virus replicated and induced both
virus-specific neutralizing antibodies and CD8+ T cells that were
not demonstrably different from those induced by YF17D alone.
[0072] FIG. 3 shows that rYF17D/SIVGag45-269 replicates and induces
neutralizing antibodies, virus-specific CD8+ T cells, and CD8+
T-cell activation in rhesus monkeys. FIG. 3A shows the replication
of rYF17D/SIVGag45-269 during the first 10 days after vaccination
with two different doses, as measured by Q-PCR using YF17D-specific
primers. FIG. 3B shows the titer of neutralizing antibodies
determined at 2 and 5 weeks after vaccination with
rYF17D/SIVGag45-269. In FIG. 3C, fresh PBMC from vaccinees
(100,00[sic] cells/well) were used in ELISPOT-IFN-.gamma. assays to
assess T-cell responses against the YF17D vector and SIV Gag
(45-269) insert. YF17D-specific responses were measured using the
same epitopes described in FIG. 2. For SIV Gag-specific responses,
6 sets of peptides of 15 mer and with 11 mer overlapping were used,
covering the whole extension of Gag 45-269 insert of SIVmac239. In
addition, Mamu-A*01-restricted responses against the dominant
GagCM9181-189 and subdominant GagQI9254-262 epitopes were measured.
The peak of CD8+ T-cell responses in r04091 is depicted at 10 days
after the second administration of rYF17D/SIVGag
(2.0.times.10.sup.5 PFU), which took place on day 28 after the
initial vaccination. FIG. 3D shows the expression kinetics of Ki-67
and Bcl-2 in CD8+ T cells after vaccination with
rYF17D/SIVGag45-269, as described in FIG. 2.
Example 4
Immunization of Rhesus Monkeys with an Initial Dose (Prime) of
Recombinant Mycobacterium bovis BCG Followed by a Booster Dose
("boost") of rYF17D/SIVGag45-269 Virus
[0073] Most viral vectors are usually more efficient after an
initial dose [prime] with DNA or rBCG [18, 19, 20, 21]. Thus, two
monkeys that had been primed with rBCG expressing SIV proteins
(FIG. 4A) were given a booster dose. No SIV-specific response was
detected after either of the two priming rBCG vaccinations.
Unfortunately, only a low level of rYF17D/SIVGag45-269 replication
was observed at day 5 after vaccination in one of the rBCG-primed
animals (r01108, 7 copies/mL; FIG. 4B). However, both animals
generated neutralizing antibodies at 2 weeks after vaccination
(FIG. 4C). Fortunately, high-frequency CD8+ T-cell responses were
detected in the Mamu-A*01-positive monkey (r01056) after a booster
dose with rYF17D/SIVGag45-269 (FIGS. 4D and 4E). The booster dose
induced massive activation of CD8+ T cells in the
Mamu-A*01-positive animal r01056, peaking at 35% at 17 days after
vaccination (FIGS. 4D and 4E). A frequency of 3.5% of the CD8+ T
cells of this animal responded to the Mamu-A*01 GagCM9181-189
epitope at day 17 after vaccination (FIGS. 4D and 4E). These CD8+ T
cells synthesized IFN .gamma., TNF.alpha., MIP-1.beta. and
degranulated (FIG. 4E and data not shown).
[0074] Thus, an rBCG prime followed by a recombinant yellow fever
17D boost induced polyfunctional antigen-specific CD8+ T cells.
[0075] Vaccine-induced CD8+ T cells may be central memory T cells
(TCM) or effector memory T cells (TEM). These two subsets of CD8+ T
cells differ in function and surface markers [22]. Repeated
boosting drives CD8+ T cells toward the TEM subset [22].
Consequently, we evaluated whether an initial dose of rBCG followed
by a booster dose of rYF17D/SIVGag45-269 induced TCM or TEM CD8+ T
cells. Staining revealed that the SIV-specific CD8+ T cells were
largely TEM cells since most of them were CD28+(FIG. 5A). It was
recently suggested that TEM cells residing in mucosas can
effectively control infection after a low-dose challenge with
SIVmac239 [23].
[0076] After that, rYF17D/SIVGag45-269-induced CD8+ T cells were
tested so as to determine if they could recognize virally infected
CD8+ T cells. It was shown that CD8+ T cells stain for tetramers
and produce cytokines after stimulation with synthetic peptides.
Nevertheless, none of these assays determines whether these CD8+ T
cells may recognize SIV-infected cells and reduce viral
replication. Thus, a recently developed assay [24] was used to
determine whether vaccine-induced CD8+ T cells can reduce viral
replication in CD4+ T cells. Sorted tetramer(-) (GagCM9181-189)
lymphocytes were incubated for 48 hours with SIVmac239-infected
CD4+ T cells that expressed Mamu-A*01. The number of CD4+ T cells
that expressed SIV Gag and the quantity of virus in the culture
supernatant were assessed. Vaccine-induced CD8+ T cells reduced
viral replication to the same extent as that seen with purified
SIV-specific CD8+ T cells from three SIVmac239-infected rhesus
monkeys, including an elite controller rhesus monkey 95061 (FIG.
5B).
[0077] Thus, in one aspect of the methods disclosed here, it is
important that the rBCG has induced a high-frequency CD8+ T-cell
response after a booster dose of rYF17D/SIVGag45-269. These CD8+ T
cells reached frequencies that were similar to those induced by an
initial dose of rBCG followed by a booster dose of Ad5 [19]. Even
without the benefit of the initial dose of rBCG, the levels of CD8+
T cells induced by a single rYF17D/SIVGag45-269 vaccination were
equivalent to those induced by our best SIV vaccine, SIVmac239ANef.
Recombinant YF17D generated an average of 195 (range of 100-750)
SFC/10 6 [sic] PBMC (N=6), whereas SIVmac239ANef induced an average
of 238 (range of 150-320) SFC/10.sup.6 PBMC (N=3) [25]. It is also
possible that any YF17D/HIV recombinants would likely replicate
better in humans than in rhesus monkeys, thus inducing more robust
immune responses. The rBCG was shown to be more effective in humans
[26, 27, 28, 29] and may be more useful for inducing T-cell
responses in humans than it has been in our limited study with
rhesus monkeys.
[0078] FIG. 4 shows that vaccination with rYF17D/SIVGag45-269
induced a robust expansion of Gag-specific responses in an
rBCG-primed monkey. FIG. 4A shows the vaccination scheme. Two
rhesus monkeys were immunized with rBCG intradermally (i.d.)
(2.times.10.sup.5 CFU), rBCG orally (10.sup.9 CFU), and
rYF17D/SIVGag45-269 subcutaneously (2.times.10.sup.5 PFU) at
6-month intervals. The rBCG was engineered to express 18 minigenes
containing sequences of Gag, Vif, Nef, Ver, and Tat from SIVmac239.
(See Tables 2-6, Proteins 1(a), 2(a), 3(a), 4(a), 6(a), 8(a),
12(a), 13(a), 14(a), 15(a), 17(a), 18 (a), 19(a), 20 (a), 21 (a),
22 (a), 23(a), e 24(a)). FIG. 4B shows the replication of
rYF17D/SIVGag45-269 during the first 10 days after vaccination, as
measured by Q-PCR using YF17D-specific primers. FIG. 4C shows the
titer of neutralizing antibodies determined at 2 and 5 weeks after
vaccination with rYF17D/SIVGag45-269. FIG. 4D shows the kinetics of
CD8+ T-cell activation (as described in FIG. 1) and expansion of
CM9181-189-specific CD8+ T cells in r01056 after vaccination with
rYF17D/SIVGag45-269. In FIG. 4E, it can be seen that vaccination
with rYF17D/SIVGag45-269 induces robust CD8+ T-cell responses
against GagCM9181-189 in r01056. CD8+ T-cell activation
(Ki-67+/Bcl-2-[sic]) for baseline and day 13 are shown.
GagCM9181-189-specific responses were measured by tetramer
staining, intracellular cytokine staining with antibodies against
MIP-1.beta. and IFN-.gamma., and ELISPOT-IFN-.gamma..
[0079] FIG. 5 illustrates that rYF17D/SIVGag45-269 vaccination of
r01056 provides effector memory GagCM9181-189-specific CD8+ T cells
that suppresses viral replication in CD4+targets. FIG. 5A shows the
frequency of tetramer-positive GagCM9181-189-specific CD8+ T cells
in r01056 gated on CD3+CD8+lymphocytes. In FIG. 5B, CD28 and CD95
expression profiles of tetramer-positive cells show a polarized
effector memory phenotype. FIG. 5C shows that ex vivo
GagCM9181-189-specific CD8+ T cells from r01056 inhibit viral
replication from SIVmac239-infected CD4+ T cells.
GagCM9181-189-specific CD8+ T cells of three SIV-infected
Manu-A*01-positive animals and r01056 vaccinated with
rYF17D/SIVGag45-269 were tested for their ability to suppress viral
replication from SIV-infected CD4+ T cells [24].
[0080] Forty-eight hours after the incubation of various samples
containing different ratios of SIV-infected CD4+ T cells and
GagCM9191-189-specific CD8+ T cells, the supernatant of these
samples was removed and measured for viral RNA copies per
milliliter by Q-PCR. No suppression was observed when effectors
were incubated with CD4+ targets from Manu-A*01-negative animals.
Rh2021 was infected with SIVmac239 (viral load .about.10.sup.5 vRNA
copies/mL) containing mutations in 8 Manu-B*08-restricted epitopes
as part of another study [30]. R01080 was vaccinated with a DNA/Ad5
regimen expressing gag, Rev, Tat and Nef, and later infected with
SIVmac239 (viral load .about.10.sup.3 vRNA copies/mL) [31]. R95061
was vaccinated with a DNA/MVA regimen containing Gag CM9181-189 and
was later challenged with SIVmac239 (undetectable viral load)
[32].
[0081] From the facts exposed above, one may conclude that the
attenuated yellow fever vaccine virus 17D expressing SIVmac239 Gag,
as described herein, provided excellent immunization responses.
[0082] The material above is illustrative of the present invention,
and is not intended to limit the scope of the claimed subject
matter. The invention is defined by the claims below.
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Sequence CWU 1
1
91510PRTSimian immunodeficiency virusmisc_feature(489)..(489)Xaa
can be any naturally occurring amino acid 1Met Gly Ala Arg Asn Ser
Val Leu Ser Gly Lys Lys Ala Asp Glu Leu1 5 10 15Glu Lys Ile Arg Leu
Arg Pro Asn Gly Lys Lys Lys Tyr Met Leu Lys 20 25 30His Val Val Trp
Ala Ala Asn Glu Leu Asp Arg Phe Gly Leu Ala Glu 35 40 45Ser Leu Leu
Glu Asn Lys Glu Gly Cys Gln Lys Ile Leu Ser Val Leu 50 55 60Ala Pro
Leu Val Pro Thr Gly Ser Glu Asn Leu Lys Ser Leu Tyr Asn65 70 75
80Thr Val Cys Val Ile Trp Cys Ile His Ala Glu Glu Lys Val Lys His
85 90 95Thr Glu Glu Ala Lys Gln Ile Val Gln Arg His Leu Val Val Glu
Thr 100 105 110Gly Thr Thr Glu Thr Met Pro Lys Thr Ser Arg Pro Thr
Ala Pro Ser 115 120 125Ser Gly Arg Gly Gly Asn Tyr Pro Val Gln Gln
Ile Gly Gly Asn Tyr 130 135 140Val His Leu Pro Leu Ser Pro Arg Thr
Leu Asn Ala Trp Val Lys Leu145 150 155 160Ile Glu Glu Lys Lys Phe
Gly Ala Glu Val Val Pro Gly Phe Gln Ala 165 170 175Leu Ser Glu Gly
Cys Thr Pro Tyr Asp Ile Asn Gln Met Leu Asn Cys 180 185 190Val Gly
Asp His Gln Ala Ala Met Gln Ile Ile Arg Asp Ile Ile Asn 195 200
205Glu Glu Ala Ala Asp Trp Asp Leu Gln His Pro Gln Pro Ala Pro Gln
210 215 220Gln Gly Gln Leu Arg Glu Pro Ser Gly Ser Asp Ile Ala Gly
Thr Thr225 230 235 240Ser Ser Val Asp Glu Gln Ile Gln Trp Met Tyr
Arg Gln Gln Asn Pro 245 250 255Ile Pro Val Gly Asn Ile Tyr Arg Arg
Trp Ile Gln Leu Gly Leu Gln 260 265 270Lys Cys Val Arg Met Tyr Asn
Pro Thr Asn Ile Leu Asp Val Lys Gln 275 280 285Gly Pro Lys Glu Pro
Phe Gln Ser Tyr Val Asp Arg Phe Tyr Lys Ser 290 295 300Leu Arg Ala
Glu Gln Thr Asp Ala Ala Val Lys Asn Trp Met Thr Gln305 310 315
320Thr Leu Leu Ile Gln Asn Ala Asn Pro Asp Cys Lys Leu Val Leu Lys
325 330 335Gly Leu Gly Val Asn Pro Thr Leu Glu Glu Met Leu Thr Ala
Cys Gln 340 345 350Gly Val Gly Gly Pro Gly Gln Lys Ala Arg Leu Met
Ala Glu Ala Leu 355 360 365Lys Glu Ala Leu Ala Pro Val Pro Ile Pro
Phe Ala Ala Ala Gln Gln 370 375 380Arg Gly Pro Arg Lys Thr Ile Lys
Cys Trp Asn Cys Gly Lys Glu Gly385 390 395 400His Ser Ala Arg Gln
Cys Arg Ala Pro Arg Arg Gln Gly Cys Trp Lys 405 410 415Cys Gly Lys
Met Asp His Val Met Ala Lys Cys Pro Asp Arg Gln Ala 420 425 430Gly
Phe Leu Gly Leu Gly Pro Trp Gly Lys Lys Pro Arg Asn Phe Pro 435 440
445Met Ala Gln Val His Gln Gly Leu Met Pro Thr Ala Pro Pro Glu Asp
450 455 460Pro Ala Val Asp Leu Leu Lys Asn Tyr Met Gln Leu Gly Lys
Gln Gln465 470 475 480Arg Glu Lys Gln Arg Glu Ser Arg Xaa Lys Pro
Tyr Lys Glu Val Thr 485 490 495Glu Asp Leu Leu His Leu Asn Ser Leu
Phe Gly Gly Asp Gln 500 505 5102307PRTSimian immunodeficiency virus
2Asp Gln Gly Ser Ala Ile Asn Phe Gly Arg Gly Leu Ala Glu Ser Leu1 5
10 15Leu Glu Asn Lys Glu Gly Cys Gln Lys Ile Leu Ser Val Leu Ala
Pro 20 25 30Leu Val Pro Thr Gly Ser Glu Asn Leu Lys Ser Leu Tyr Asn
Thr Val 35 40 45Cys Val Ile Trp Cys Ile His Ala Glu Glu Lys Val Lys
His Thr Glu 50 55 60Glu Ala Lys Gln Ile Val Gln Arg His Leu Val Val
Glu Thr Gly Thr65 70 75 80Thr Glu Thr Met Pro Lys Thr Ser Arg Pro
Thr Ala Pro Ser Ser Gly 85 90 95Arg Gly Gly Asn Tyr Pro Val Gln Gln
Ile Gly Gly Asn Tyr Val His 100 105 110Leu Pro Leu Ser Pro Arg Thr
Leu Asn Ala Trp Val Lys Leu Ile Glu 115 120 125Glu Lys Lys Phe Gly
Ala Glu Val Val Pro Gly Phe Gln Ala Leu Ser 130 135 140Glu Gly Cys
Thr Pro Tyr Asp Ile Asn Gln Met Leu Asn Cys Val Gly145 150 155
160Asp His Gln Ala Ala Met Gln Ile Ile Arg Asp Ile Ile Asn Glu Glu
165 170 175Ala Ala Asp Trp Asp Leu Gln His Pro Gln Pro Ala Pro Gln
Gln Gly 180 185 190Gln Leu Arg Glu Pro Ser Gly Ser Asp Ile Ala Gly
Thr Thr Ser Ser 195 200 205Val Asp Glu Gln Ile Gln Trp Met Tyr Arg
Gln Gln Asn Pro Ile Pro 210 215 220Val Gly Asn Ile Tyr Arg Arg Trp
Ile Gln Leu Lys Glu Ser Ser Ile225 230 235 240Gly Thr Ser Leu Gly
Lys Ala Val His Gln Val Phe Gly Ser Val Tyr 245 250 255Thr Thr Met
Phe Gly Gly Val Ser Trp Met Ile Arg Ile Leu Ile Gly 260 265 270Phe
Leu Val Leu Trp Ile Gly Thr Asn Ser Arg Asn Thr Ser Met Ala 275 280
285Met Thr Cys Ile Ala Val Gly Gly Ile Thr Leu Phe Leu Gly Phe Thr
290 295 300Val Gly Ala305310PRTYellow fever virus 3Asp Gln Gly Ser
Ala Ile Asn Phe Gly Arg1 5 1046PRTYellow fever virus 4Lys Glu Ser
Ser Ile Gly1 5566PRTDengue virus type 4 5Thr Ser Leu Gly Lys Ala
Val His Gln Val Phe Gly Ser Val Tyr Thr1 5 10 15Thr Met Phe Gly Gly
Val Ser Trp Met Ile Arg Ile Leu Ile Gly Phe 20 25 30Leu Val Leu Trp
Ile Gly Thr Asn Ser Arg Asn Thr Ser Met Ala Met 35 40 45Thr Cys Ile
Ala Val Gly Gly Ile Thr Leu Phe Leu Gly Phe Thr Val 50 55 60Gly
Ala656924DNASimian immunodeficiency virus 6gatcaaggaa gcgccatcaa
cttcggccga ggactggctg aatcactgct ggaaaacaaa 60gaaggatgtc agaaaatcct
gtcagtgctg gctccactgg tgccaacagg atcagaaaac 120ctgaagtcac
tgtacaacac agtgtgtgtg atctggtgta tccatgctga agaaaaagtg
180aagcatacag aagaggccaa gcagatcgtg cagaggcatc tggtggtgga
aacagggaca 240acagaaacaa tgccaaagac atcaaggcca acagctccat
catcaggaag ggggggaaac 300tacccagtgc agcagatcgg agggaactac
gtgcatctgc cactgtcacc aaggacactg 360aacgcttggg tgaaactgat
cgaagaaaag aaatttggag ctgaagtggt gccaggattt 420caggctctgt
cagaaggatg tacaccatac gacatcaacc agatgctgaa ctgtgtggga
480gatcatcagg ctgctatgca gatcattagg gacatcatca acgaagaagc
tgcagattgg 540gatctgcaac atccacagcc agctccacag cagggacagc
tgagggaacc atcaggatca 600gacattgctg gaacaacatc atcagtggac
gaacagatcc agtggatgta caggcagcag 660aacccaatcc cagtgggaaa
catctacaga agatggatcc agctgaaaga gggaagctca 720ataggaacat
cattgggaaa ggctgtgcac caggtttttg gaagtgtgta tacaaccatg
780tttggaggag tctcatggat gattagaatc ctaattgggt tcttagtgtt
gtggattggc 840acgaactcca ggaacacttc aatggctatg acgtgcatag
ctgttggagg aatcactctg 900tttctgggct tcacagttgg cgcc
924711785DNASimian immunodeficiency virus 7agtaaatcct gtgtgctaat
tgaggtgcat tggtctgcaa atcgagttgc taggcaataa 60acacatttgg attaatttta
atcgttcgtt gagcgattag cagagaactg accagaacat 120gtctggtcgt
aaagctcagg gaaaaaccct gggcgtcaat atggtacgac gaggagttcg
180ctccttgtca aacaaaataa aacaaaaaac aaaacaaatt ggaaacagac
ctggaccttc 240aagaggtgtt caaggattta tctttttctt tttgttcaac
attttgactg gaaaaaagat 300cacagcccac ctaaagaggt tgtggaaaat
gctggaccca agacaaggct tggctgttct 360aaggaaagtc aagagagtgg
tggccagttt gatgagagga ttgtcctcaa ggaaacgccg 420ttcccatgat
gttctgactg tgcaattcct aattttggga atgctgttga tgaccggtgg
480agtgaccttg gtgcggaaaa acagatggtt gctcctaaat gtgacatctg
aggacctcgg 540gaaaacattc tctgtgggca caggcaactg cacaacaaac
attttggaag ccaagtactg 600gtgcccagac tcaatggaat acaactgtcc
caatctcagt ccaagagagg agccagatga 660cattgattgc tggtgctatg
gggtggaaaa cgttagagtc gcatatggta agtgtgactc 720agcaggcagg
tctaggaggt caagaagggc cattgacttg cctacgcatg aaaaccatgg
780tttgaagacc cggcaagaaa aatggatgac tggaagaatg ggtgaaaggc
aactccaaaa 840gattgagaga tggttcgtga ggaacccctt ttttgcagtg
acggctctga ccattgccta 900ccttgtggga agcaacatga cgcaacgagt
cgtgattgcc ctactggtct tggctgttgg 960tccggcctac tcagctcact
gcattggaat tactgacagg gatttcattg agggggtgca 1020tggaggaact
tgggtttcag ctaccctgga gcaagacaag tgtgtcactg ttatggcccc
1080tgacaagcct tcattggaca tctcactaga gacagtagcc attgatagac
ctgctgaggc 1140gaggaaagtg tgttacaatg cagttctcac tcatgtgaag
attaatgaca agtgccccag 1200cactggagag gcccacctag ctgaagagaa
cgaaggggac aatgcgtgca agcgcactta 1260ttctgataga ggctggggca
atggctgtgg cctatttggg aaagggagca ttgtggcatg 1320cgccaaattc
acttgtgcca aatccatgag tttgtttgag gttgatcaga ccaaaattca
1380gtatgtcatc agagcacaat tgcatgtagg ggccaagcag gaaaattgga
ataccagcat 1440taagactctc aagtttgatg ccctgtcagg ctcccaggaa
gtcgagttca ttgggtatgg 1500aaaagctaca ctggaatgcc aggtgcaaac
tgcggtggac tttggtaaca gttacatcgc 1560tgagatggaa acagagagct
ggatagtgga cagacagtgg gcccaggact tgaccctgcc 1620atggcagagt
ggaagtggcg gggtgtggag agagatgcat catcttgtcg aatttgaacc
1680tccgcatgcc gccactatca gagtactggc cctgggaaac caggaaggct
ccttgaaaac 1740agctcttact ggcgcaatga gggttacaaa ggacacaaat
gacaacaacc tttacaaact 1800acatggtgga catgtttctt gcagagtgaa
attgtcagct ttgacactca aggggacatc 1860ctacaaaata tgcactgaca
aaatgttttt tgtcaagaac ccaactgaca ctggccatgg 1920cactgttgtg
atgcaggtga aagtgtcaaa aggaaccccc tgcaggattc cagtgatagt
1980agctgatgat cttacagcgg caatcaataa aggcattttg gttacagtta
accccatcgc 2040ctcaaccaat gatgatgaag tgctgattga ggtgaaccca
ccttttggag acagctacat 2100tatcgttggg agaggagatt cacgtctcac
ttaccagtgg cacaaagagg gaagctcaat 2160aggaaagttg ttcactcaga
ccatgaaagg cgtggaacgc ctggccgtca tgggagacac 2220cgcctgggat
ttcagctccg ctggagggtt cttcacttcg gttgggaaag gaattcatac
2280ggtgtttggc tctgcctttc aggggctatt tggcggcttg aactggataa
caaaggtcat 2340catgggggcg gtactcatat gggttggcat caacacaaga
aacatgacaa tgtccatgag 2400catgatcttg gtaggagtga tcatgatgtt
tttgtctcta ggagttgggg cggatcaagg 2460aagcgccatc aacttcggcc
gaggactggc tgaatcactg ctggaaaaca aagaaggatg 2520tcagaaaatc
ctgtcagtgc tggctccact ggtgccaaca ggatcagaaa acctgaagtc
2580actgtacaac acagtgtgtg tgatctggtg tatccatgct gaagaaaaag
tgaagcatac 2640agaagaggcc aagcagatcg tgcagaggca tctggtggtg
gaaacaggga caacagaaac 2700aatgccaaag acatcaaggc caacagctcc
atcatcagga agggggggaa actacccagt 2760gcagcagatc ggagggaact
acgtgcatct gccactgtca ccaaggacac tgaacgcttg 2820ggtgaaactg
atcgaagaaa agaaatttgg agctgaagtg gtgccaggat ttcaggctct
2880gtcagaagga tgtacaccat acgacatcaa ccagatgctg aactgtgtgg
gagatcatca 2940ggctgctatg cagatcatta gggacatcat caacgaagaa
gctgcagatt gggatctgca 3000acatccacag ccagctccac agcagggaca
gctgagggaa ccatcaggat cagacattgc 3060tggaacaaca tcatcagtgg
acgaacagat ccagtggatg tacaggcagc agaacccaat 3120cccagtggga
aacatctaca gaagatggat ccagctgaaa gagggaagct caataggaac
3180atcattggga aaggctgtgc accaggtttt tggaagtgtg tatacaacca
tgtttggagg 3240agtctcatgg atgattagaa tcctaattgg gttcttagtg
ttgtggattg gcacgaactc 3300caggaacact tcaatggcta tgacgtgcat
agctgttgga ggaatcactc tgtttctggg 3360cttcacagtt ggcgccgatc
aaggatgcgc catcaacttt ggcaagagag agctcaagtg 3420cggagatggt
atcttcatat ttagagactc tgatgactgg ctgaacaagt actcatacta
3480tccagaagat cctgtgaagc ttgcatcaat agtgaaagcc tctttcgaag
aagggaagtg 3540tggcctaaat tcagttgact cccttgagca tgagatgtgg
agaagcaggg cagatgagat 3600taataccatt tttgaggaaa acgaggtgga
catttctgtt gtcgtgcagg atccaaagaa 3660tgtttaccag agaggaactc
atccattttc cagaattcgg gatggtctgc agtatggttg 3720gaagacttgg
ggtaagaacc ttgtgttctc cccagggagg aagaatggaa gcttcatcat
3780agatggaaag tccaggaaag aatgcccgtt ttcaaaccgg gtctggaatt
ctttccagat 3840agaggagttt gggacgggag tgttcaccac acgcgtgtac
atggacgcag tctttgaata 3900caccatagac tgcgatggat ctatcttggg
tgcagcggtg aacggaaaaa agagtgccca 3960tggctctcca acattttgga
tgggaagtca tgaagtaaat gggacatgga tgatccacac 4020cttggaggca
ttagattaca aggagtgtga gtggccactg acacatacga ttggaacatc
4080agttgaagag agtgaaatgt tcatgccgag atcaatcgga ggcccagtta
gctctcacaa 4140tcatatccct ggatacaagg ttcagacgaa cggaccttgg
atgcaggtac cactagaagt 4200gaagagagaa gcttgcccag ggactagcgt
gatcattgat ggcaactgtg atggacgggg 4260aaaatcaacc agatccacca
cggatagcgg gaaagttatt cctgaatggt gttgccgctc 4320ctgcacaatg
ccgcctgtga gcttccatgg tagtgatggg tgttggtatc ccatggaaat
4380taggccaagg aaaacgcatg aaagccatct ggtgcgctcc tgggttacag
ctggagaaat 4440acatgctgtc ccttttggtt tggtgagcat gatgatagca
atggaagtgg tcctaaggaa 4500aagacaggga ccaaagcaaa tgttggttgg
aggagtagtg ctcttgggag caatgctggt 4560cgggcaagta actctccttg
atttgctgaa actcacagtg gctgtgggat tgcatttcca 4620tgagatgaac
aatggaggag acgccatgta tatggcgttg attgctgcct tttcaatcag
4680accagggctg ctcatcggct ttgggctcag gaccctatgg agccctcggg
aacgccttgt 4740gctgacccta ggagcagcca tggtggagat tgccttgggt
ggcgtgatgg gcggcctgtg 4800gaagtatcta aatgcagttt ctctctgcat
cctgacaata aatgctgttg cttctaggaa 4860agcatcaaat accatcttgc
ccctcatggc tctgttgaca cctgtcacta tggctgaggt 4920gagacttgcc
gcaatgttct tttgtgccat ggttatcata ggggtccttc accagaattt
4980caaggacacc tccatgcaga agactatacc tctggtggcc ctcacactca
catcttacct 5040gggcttgaca caaccttttt tgggcctgtg tgcatttctg
gcaacccgca tatttgggcg 5100aaggagtatc ccagtgaatg aggcactcgc
agcagctggt ctagtgggag tgctggcagg 5160actggctttt caggagatgg
agaacttcct tggtccgatt gcagttggag gactcctgat 5220gatgctggtt
agcgtggctg ggagggtgga tgggctagag ctcaagaagc ttggtgaagt
5280ttcatgggaa gaggaggcgg agatcagcgg gagttccgcc cgctatgatg
tggcactcag 5340tgaacaaggg gagttcaagc tgctttctga agagaaagtg
ccatgggacc aggttgtgat 5400gacctcgctg gccttggttg gggctgccct
ccatccattt gctcttctgc tggtccttgc 5460tgggtggctg tttcatgtca
ggggagctag gagaagtggg gatgtcttgt gggatattcc 5520cactcctaag
atcatcgagg aatgtgaaca tctggaggat gggatttatg gcatattcca
5580gtcaaccttc ttgggggcct cccagcgagg agtgggagtg gcacagggag
gggtgttcca 5640cacaatgtgg catgtcacaa gaggagcttt ccttgtcagg
aatggcaaga agttgattcc 5700atcttgggct tcagtaaagg aagaccttgt
cgcctatggt ggctcatgga agttggaagg 5760cagatgggat ggagaggaag
aggtccagtt gatcgcggct gttccaggaa agaacgtggt 5820caacgtccag
acaaaaccga gcttgttcaa agtgaggaat gggggagaaa tcggggctgt
5880cgctcttgac tatccgagtg gcacttcagg atctcctatt gttaacagga
acggagaggt 5940gattgggctg tacggcaatg gcatccttgt cggtgacaac
tccttcgtgt ccgccatatc 6000ccagactgag gtgaaggaag aaggaaagga
ggagctccaa gagatcccga caatgctaaa 6060gaaaggaatg acaactgtcc
ttgattttca tcctggagct gggaagacaa gacgtttcct 6120cccacagatc
ttggccgagt gcgcacggag acgcttgcgc actcttgtgt tggcccccac
6180cagggttgtt ctttctgaaa tgaaggaggc ttttcacggc ctggacgtga
aattccacac 6240acaggctttt tccgctcacg gcagcgggag agaagtcatt
gatgccatgt gccatgccac 6300cctaacttac aggatgttgg aaccaactag
ggttgttaac tgggaagtga tcattatgga 6360tgaagcccat tttttggatc
cagctagcat agccgctaga ggttgggcag cgcacagagc 6420tagggcaaat
gaaagtgcaa caatcttgat gacagccaca ccgcctggga ctagtgatga
6480atttccacat tcaaatggtg aaatagaaga tgttcaaacg gacataccca
gtgagccctg 6540gaacacaggg catgactgga tcctggctga caaaaggccc
acggcatggt tccttccatc 6600catcagagct gcaaatgtca tggctgcctc
tttgcgtaag gctggaaaga gtgtggtggt 6660cctgaacagg aaaacctttg
agagagaata ccccacgata aagcagaaga aacctgactt 6720tatattggcc
actgacatag ctgaaatggg agccaacctt tgcgtggagc gagtgctgga
6780ttgcaggacg gcttttaagc ctgtgcttgt ggatgaaggg aggaaggtgg
caataaaagg 6840gccacttcgt atctccgcat cctctgctgc tcaaaggagg
gggcgcattg ggagaaatcc 6900caacagagat ggagactcat actactattc
tgagcctaca agtgaaaata atgcccacca 6960cgtctgctgg ttggaggcct
caatgctctt ggacaacatg gaggtgaggg gtggaatggt 7020cgccccactc
tatggcgttg aaggaactaa aacaccagtt tcccctggtg aaatgagact
7080gagggatgac cagaggaaag tcttcagaga actagtgagg aattgtgacc
tgcccgtttg 7140gctttcgtgg caagtggcca aggctggttt gaagacgaat
gatcgtaagt ggtgttttga 7200aggccctgag gaacatgaga tcttgaatga
cagcggtgaa acagtgaagt gcagggctcc 7260tggaggagca aagaagcctc
tgcgcccaag gtggtgtgat gaaagggtgt catctgacca 7320gagtgcgctg
tctgaattta ttaagtttgc tgaaggtagg aggggagctg ctgaagtgct
7380agttgtgctg agtgaactcc ctgatttcct ggctaaaaaa ggtggagagg
caatggatac 7440catcagtgtg ttcctccact ctgaggaagg ctctagggct
taccgcaatg cactatcaat 7500gatgcctgag gcaatgacaa tagtcatgct
gtttatactg gctggactac tgacatcggg 7560aatggtcatc tttttcatgt
ctcccaaagg catcagtaga atgtctatgg cgatgggcac 7620aatggccggc
tgtggatatc tcatgttcct tggaggcgtc aaacccactc acatctccta
7680tgtcatgctc atattctttg tcctgatggt ggttgtgatc cccgagccag
ggcaacaaag 7740gtccatccaa gacaaccaag tggcatacct cattattggc
atcctgacgc tggtttcagc 7800ggtggcagcc aacgagctag gcatgctgga
gaaaaccaaa gaggacctct ttgggaagaa 7860gaacttaatt ccatctagtg
cttcaccctg gagttggccg gatcttgacc tgaagccagg 7920agctgcctgg
acagtgtacg ttggcattgt tacaatgctc tctccaatgt tgcaccactg
7980gatcaaagtc gaatatggca acctgtctct gtctggaata gcccagtcag
cctcagtcct 8040ttctttcatg gacaagggga taccattcat gaagatgaat
atctcggtca taatgctgct 8100ggtcagtggc tggaattcaa taacagtgat
gcctctgctc tgtggcatag ggtgcgccat 8160gctccactgg tctctcattt
tacctggaat caaagcgcag cagtcaaagc ttgcacagag 8220aagggtgttc
catggcgttg ccaagaaccc tgtggttgat gggaatccaa cagttgacat
8280tgaggaagct cctgaaatgc ctgcccttta tgagaagaaa ctggctctat
atctccttct 8340tgctctcagc ctagcttctg ttgccatgtg cagaacgccc
ttttcattgg ctgaaggcat
8400tgtcctagca tcagctgcct tagggccgct catagaggga aacaccagcc
ttctttggaa 8460tggacccatg gctgtctcca tgacaggagt catgaggggg
aatcactatg cttttgtggg 8520agtcatgtac aatctatgga agatgaaaac
tggacgccgg gggagcgcga atggaaaaac 8580tttgggtgaa gtctggaaga
gggaactgaa tctgttggac aagcgacagt ttgagttgta 8640taaaaggacc
gacattgtgg aggtggatcg tgatacggca cgcaggcatt tggccgaagg
8700gaaggtggac accggggtgg cggtctccag ggggaccgca aagttaaggt
ggttccatga 8760gcgtggctat gtcaagctgg aaggtagggt gattgacctg
gggtgtggcc gcggaggctg 8820gtgttactac gctgctgcgc aaaaggaagt
gagtggggtc aaaggattta ctcttggaag 8880agacggccat gagaaaccca
tgaatgtgca aagtctggga tggaacatca tcaccttcaa 8940ggacaaaact
gatatccacc gcctagaacc agtgaaatgt gacacccttt tgtgtgacat
9000tggagagtca tcatcgtcat cggtcacaga gggggaaagg accgtgagag
ttcttgatac 9060tgtagaaaaa tggctggctt gtggggttga caacttctgt
gtgaaggtgt tagctccata 9120catgccagat gttcttgaga aactggaatt
gctccaaagg aggtttggcg gaacagtgat 9180caggaaccct ctctccagga
attccactca tgaaatgtac tacgtgtctg gagcccgcag 9240caatgtcaca
tttactgtga accaaacatc ccgcctcctg atgaggagaa tgaggcgtcc
9300aactggaaaa gtgaccctgg aggctgacgt catcctccca attgggacac
gcagtgttga 9360gacagacaag ggacccctgg acaaagaggc catagaagaa
agggttgaga ggataaaatc 9420tgagtacatg acctcttggt tttatgacaa
tgacaacccc tacaggacct ggcactactg 9480tggctcctat gtcacaaaaa
cctcaggaag tgcggcgagc atggtaaatg gtgttattaa 9540aattctgaca
tatccatggg acaggataga ggaggtcacc agaatggcaa tgactgacac
9600aacccctttt ggacagcaaa gagtgtttaa agaaaaagtt gacaccagag
caaaggatcc 9660accagcggga actaggaaga tcatgaaagt tgtcaacagg
tggctgttcc gccacctggc 9720cagagaaaag agccccagac tgtgcacaaa
ggaagaattt attgcaaaag tccgaagtca 9780tgcagccatt ggagcttacc
tggaagaaca agaacagtgg aagactgcca atgaggctgt 9840ccaagaccca
aagttctggg aactggtgga tgaagaaagg aagctgcacc aacaaggcag
9900gtgtcggact tgtgtgtaca acatgatggg gaaaagagag aagaagctgt
cagagtttgg 9960gaaagcaaag ggaagccgtg ccatatggta tatgtggctg
ggagcgcggt atcttgagtt 10020tgaggccctg ggattcctga atgaggacca
ttgggcttcc agggaaaact caggaggagg 10080agtggaaggc attggcttac
aatacctagg atatgtgatc agagacctgg ctgcaatgga 10140tggtggtgga
ttctacgcgg atgacaccgc tggatgggac acgcgcatca cagaggcaga
10200ccttgatgat gaacaggaga tcttgaacta catgagccca catcacaaaa
aactggcaca 10260agcagtgatg gaaatgacat acaagaacaa agtggtgaaa
gtgttgagac cagccccagg 10320agggaaagcc tacatggatg tcataagtcg
acgagaccag agaggatccg ggcaggtagt 10380gacttatgct ctgaacacca
tcaccaactt gaaagtccaa ttgatcagaa tggcagaagc 10440agagatggtg
atacatcacc aacatgttca agattgtgat gaatcagttc tgaccaggct
10500ggaggcatgg ctcactgagc acggatgtaa cagactgaag aggatggcgg
tgagtggaga 10560cgactgtgtg gtccggccca tcgatgacag gttcggcctg
gccctgtccc atctcaacgc 10620catgtccaag gttagaaagg acatatctga
atggcagcca tcaaaagggt ggaatgattg 10680ggagaatgtg cccttctgtt
cccaccactt ccatgaacta cagctgaagg atggcaggag 10740gattgtggtg
ccttgccgag aacaggacga gctcattggg agaggaaggg tgtctccagg
10800aaacggctgg atgatcaagg aaacagcttg cctcagcaaa gcctatgcca
acatgtggtc 10860actgatgtat tttcacaaaa gggacatgag gctactgtca
ttggctgttt cctcagctgt 10920tcccacctca tgggttccac aaggacgcac
aacatggtcg attcatggga aaggggagtg 10980gatgaccacg gaagacatgc
ttgaggtgtg gaacagagta tggataacca acaacccaca 11040catgcaggac
aagacaatgg tgaaaaaatg gagagatgtc ccttatctaa ccaagagaca
11100agacaagctg tgcggatcac tgattggaat gaccaatagg gccacctggg
cctcccacat 11160ccatttagtc atccatcgta tccgaacgct gattggacag
gagaaataca ctgactacct 11220aacagtcatg gacaggtatt ctgtggatgc
tgacctgcaa ctgggtgagc ttatctgaaa 11280caccatctaa caggaataac
cgggatacaa accacgggtg gagaaccgga ctccccacaa 11340cctgaaaccg
ggatataaac cacggctgga gaaccgggct ccgcacttaa aatgaaacag
11400aaaccgggat aaaaactacg gatggagaac cggactccac acattgagac
agaagaagtt 11460gtcagcccag aaccccacac gagttttgcc actgctaagc
tgtgaggcag tgcaggctgg 11520gacagccgac ctccaggttg cgaaaaacct
ggtttctggg acctcccacc ccagagtaaa 11580aagaacggag cctccgctac
caccctccca cgtggtggta gaaagacggg gtctagaggt 11640tagaggagac
cctccaggga acaaatagtg ggaccatatt gacgccaggg aaagaccgga
11700gtggttctct gcttttcctc cagaggtctg tgagcacagt ttgctcaaga
ataagcagac 11760ctttggatga caaacacaaa accac 1178583718PRTSimian
immunodeficiency virus 8Ser Gly Arg Lys Ala Gln Gly Lys Thr Leu Gly
Val Asn Met Val Arg1 5 10 15Arg Gly Val Arg Ser Leu Ser Asn Lys Ile
Lys Gln Lys Thr Lys Gln 20 25 30Ile Gly Asn Arg Pro Gly Pro Ser Arg
Gly Val Gln Gly Phe Ile Phe 35 40 45Phe Phe Leu Phe Asn Ile Leu Thr
Gly Lys Lys Ile Thr Ala His Leu 50 55 60Lys Arg Leu Trp Lys Met Leu
Asp Pro Arg Gln Gly Leu Ala Val Leu65 70 75 80Arg Lys Val Lys Arg
Val Val Ala Ser Leu Met Arg Gly Leu Ser Ser 85 90 95Arg Lys Arg Arg
Ser His Asp Val Leu Thr Val Gln Phe Leu Ile Leu 100 105 110Gly Met
Leu Leu Met Thr Gly Gly Val Thr Leu Val Arg Lys Asn Arg 115 120
125Trp Leu Leu Leu Asn Val Thr Ser Glu Asp Leu Gly Lys Thr Phe Ser
130 135 140Val Gly Thr Gly Asn Cys Thr Thr Asn Ile Leu Glu Ala Lys
Tyr Trp145 150 155 160Cys Pro Asp Ser Met Glu Tyr Asn Cys Pro Asn
Leu Ser Pro Arg Glu 165 170 175Glu Pro Asp Asp Ile Asp Cys Trp Cys
Tyr Gly Val Glu Asn Val Arg 180 185 190Val Ala Tyr Gly Lys Cys Asp
Ser Ala Gly Arg Ser Arg Arg Ser Arg 195 200 205Arg Ala Ile Asp Leu
Pro Thr His Glu Asn His Gly Leu Lys Thr Arg 210 215 220Gln Glu Lys
Trp Met Thr Gly Arg Met Gly Glu Arg Gln Leu Gln Lys225 230 235
240Ile Glu Arg Trp Phe Val Arg Asn Pro Phe Phe Ala Val Thr Ala Leu
245 250 255Thr Ile Ala Tyr Leu Val Gly Ser Asn Met Thr Gln Arg Val
Val Ile 260 265 270Ala Leu Leu Val Leu Ala Val Gly Pro Ala Tyr Ser
Ala His Cys Ile 275 280 285Gly Ile Thr Asp Arg Asp Phe Ile Glu Gly
Val His Gly Gly Thr Trp 290 295 300Val Ser Ala Thr Leu Glu Gln Asp
Lys Cys Val Thr Val Met Ala Pro305 310 315 320Asp Lys Pro Ser Leu
Asp Ile Ser Leu Glu Thr Val Ala Ile Asp Arg 325 330 335Pro Ala Glu
Ala Arg Lys Val Cys Tyr Asn Ala Val Leu Thr His Val 340 345 350Lys
Ile Asn Asp Lys Cys Pro Ser Thr Gly Glu Ala His Leu Ala Glu 355 360
365Glu Asn Glu Gly Asp Asn Ala Cys Lys Arg Thr Tyr Ser Asp Arg Gly
370 375 380Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser Ile Val
Ala Cys385 390 395 400Ala Lys Phe Thr Cys Ala Lys Ser Met Ser Leu
Phe Glu Val Asp Gln 405 410 415Thr Lys Ile Gln Tyr Val Ile Arg Ala
Gln Leu His Val Gly Ala Lys 420 425 430Gln Glu Asn Trp Asn Thr Ser
Ile Lys Thr Leu Lys Phe Asp Ala Leu 435 440 445Ser Gly Ser Gln Glu
Val Glu Phe Ile Gly Tyr Gly Lys Ala Thr Leu 450 455 460Glu Cys Gln
Val Gln Thr Ala Val Asp Phe Gly Asn Ser Tyr Ile Ala465 470 475
480Glu Met Glu Thr Glu Ser Trp Ile Val Asp Arg Gln Trp Ala Gln Asp
485 490 495Leu Thr Leu Pro Trp Gln Ser Gly Ser Gly Gly Val Trp Arg
Glu Met 500 505 510His His Leu Val Glu Phe Glu Pro Pro His Ala Ala
Thr Ile Arg Val 515 520 525Leu Ala Leu Gly Asn Gln Glu Gly Ser Leu
Lys Thr Ala Leu Thr Gly 530 535 540Ala Met Arg Val Thr Lys Asp Thr
Asn Asp Asn Asn Leu Tyr Lys Leu545 550 555 560His Gly Gly His Val
Ser Cys Arg Val Lys Leu Ser Ala Leu Thr Leu 565 570 575Lys Gly Thr
Ser Tyr Lys Ile Cys Thr Asp Lys Met Phe Phe Val Lys 580 585 590Asn
Pro Thr Asp Thr Gly His Gly Thr Val Val Met Gln Val Lys Val 595 600
605Ser Lys Gly Thr Pro Cys Arg Ile Pro Val Ile Val Ala Asp Asp Leu
610 615 620Thr Ala Ala Ile Asn Lys Gly Ile Leu Val Thr Val Asn Pro
Ile Ala625 630 635 640Ser Thr Asn Asp Asp Glu Val Leu Ile Glu Val
Asn Pro Pro Phe Gly 645 650 655Asp Ser Tyr Ile Ile Val Gly Arg Gly
Asp Ser Arg Leu Thr Tyr Gln 660 665 670Trp His Lys Glu Gly Ser Ser
Ile Gly Lys Leu Phe Thr Gln Thr Met 675 680 685Lys Gly Val Glu Arg
Leu Ala Val Met Gly Asp Thr Ala Trp Asp Phe 690 695 700Ser Ser Ala
Gly Gly Phe Phe Thr Ser Val Gly Lys Gly Ile His Thr705 710 715
720Val Phe Gly Ser Ala Phe Gln Gly Leu Phe Gly Gly Leu Asn Trp Ile
725 730 735Thr Lys Val Ile Met Gly Ala Val Leu Ile Trp Val Gly Ile
Asn Thr 740 745 750Arg Asn Met Thr Met Ser Met Ser Met Ile Leu Val
Gly Val Ile Met 755 760 765Met Phe Leu Ser Leu Gly Val Gly Ala Asp
Gln Gly Ser Ala Ile Asn 770 775 780Phe Gly Arg Gly Leu Ala Glu Ser
Leu Leu Glu Asn Lys Glu Gly Cys785 790 795 800Gln Lys Ile Leu Ser
Val Leu Ala Pro Leu Val Pro Thr Gly Ser Glu 805 810 815Asn Leu Lys
Ser Leu Tyr Asn Thr Val Cys Val Ile Trp Cys Ile His 820 825 830Ala
Glu Glu Lys Val Lys His Thr Glu Glu Ala Lys Gln Ile Val Gln 835 840
845Arg His Leu Val Val Glu Thr Gly Thr Thr Glu Thr Met Pro Lys Thr
850 855 860Ser Arg Pro Thr Ala Pro Ser Ser Gly Arg Gly Gly Asn Tyr
Pro Val865 870 875 880Gln Gln Ile Gly Gly Asn Tyr Val His Leu Pro
Leu Ser Pro Arg Thr 885 890 895Leu Asn Ala Trp Val Lys Leu Ile Glu
Glu Lys Lys Phe Gly Ala Glu 900 905 910Val Val Pro Gly Phe Gln Ala
Leu Ser Glu Gly Cys Thr Pro Tyr Asp 915 920 925Ile Asn Gln Met Leu
Asn Cys Val Gly Asp His Gln Ala Ala Met Gln 930 935 940Ile Ile Arg
Asp Ile Ile Asn Glu Glu Ala Ala Asp Trp Asp Leu Gln945 950 955
960His Pro Gln Pro Ala Pro Gln Gln Gly Gln Leu Arg Glu Pro Ser Gly
965 970 975Ser Asp Ile Ala Gly Thr Thr Ser Ser Val Asp Glu Gln Ile
Gln Trp 980 985 990Met Tyr Arg Gln Gln Asn Pro Ile Pro Val Gly Asn
Ile Tyr Arg Arg 995 1000 1005Trp Ile Gln Leu Lys Glu Gly Ser Ser
Ile Gly Thr Ser Leu Gly 1010 1015 1020Lys Ala Val His Gln Val Phe
Gly Ser Val Tyr Thr Thr Met Phe 1025 1030 1035Gly Gly Val Ser Trp
Met Ile Arg Ile Leu Ile Gly Phe Leu Val 1040 1045 1050Leu Trp Ile
Gly Thr Asn Ser Arg Asn Thr Ser Met Ala Met Thr 1055 1060 1065Cys
Ile Ala Val Gly Gly Ile Thr Leu Phe Leu Gly Phe Thr Val 1070 1075
1080Gly Ala Asp Gln Gly Cys Ala Ile Asn Phe Gly Lys Arg Glu Leu
1085 1090 1095Lys Cys Gly Asp Gly Ile Phe Ile Phe Arg Asp Ser Asp
Asp Trp 1100 1105 1110Leu Asn Lys Tyr Ser Tyr Tyr Pro Glu Asp Pro
Val Lys Leu Ala 1115 1120 1125Ser Ile Val Lys Ala Ser Phe Glu Glu
Gly Lys Cys Gly Leu Asn 1130 1135 1140Ser Val Asp Ser Leu Glu His
Glu Met Trp Arg Ser Arg Ala Asp 1145 1150 1155Glu Ile Asn Thr Ile
Phe Glu Glu Asn Glu Val Asp Ile Ser Val 1160 1165 1170Val Val Gln
Asp Pro Lys Asn Val Tyr Gln Arg Gly Thr His Pro 1175 1180 1185Phe
Ser Arg Ile Arg Asp Gly Leu Gln Tyr Gly Trp Lys Thr Trp 1190 1195
1200Gly Lys Asn Leu Val Phe Ser Pro Gly Arg Lys Asn Gly Ser Phe
1205 1210 1215Ile Ile Asp Gly Lys Ser Arg Lys Glu Cys Pro Phe Ser
Asn Arg 1220 1225 1230Val Trp Asn Ser Phe Gln Ile Glu Glu Phe Gly
Thr Gly Val Phe 1235 1240 1245Thr Thr Arg Val Tyr Met Asp Ala Val
Phe Glu Tyr Thr Ile Asp 1250 1255 1260Cys Asp Gly Ser Ile Leu Gly
Ala Ala Val Asn Gly Lys Lys Ser 1265 1270 1275Ala His Gly Ser Pro
Thr Phe Trp Met Gly Ser His Glu Val Asn 1280 1285 1290Gly Thr Trp
Met Ile His Thr Leu Glu Ala Leu Asp Tyr Lys Glu 1295 1300 1305Cys
Glu Trp Pro Leu Thr His Thr Ile Gly Thr Ser Val Glu Glu 1310 1315
1320Ser Glu Met Phe Met Pro Arg Ser Ile Gly Gly Pro Val Ser Ser
1325 1330 1335His Asn His Ile Pro Gly Tyr Lys Val Gln Thr Asn Gly
Pro Trp 1340 1345 1350Met Gln Val Pro Leu Glu Val Lys Arg Glu Ala
Cys Pro Gly Thr 1355 1360 1365Ser Val Ile Ile Asp Gly Asn Cys Asp
Gly Arg Gly Lys Ser Thr 1370 1375 1380Arg Ser Thr Thr Asp Ser Gly
Lys Val Ile Pro Glu Trp Cys Cys 1385 1390 1395Arg Ser Cys Thr Met
Pro Pro Val Ser Phe His Gly Ser Asp Gly 1400 1405 1410Cys Trp Tyr
Pro Met Glu Ile Arg Pro Arg Lys Thr His Glu Ser 1415 1420 1425His
Leu Val Arg Ser Trp Val Thr Ala Gly Glu Ile His Ala Val 1430 1435
1440Pro Phe Gly Leu Val Ser Met Met Ile Ala Met Glu Val Val Leu
1445 1450 1455Arg Lys Arg Gln Gly Pro Lys Gln Met Leu Val Gly Gly
Val Val 1460 1465 1470Leu Leu Gly Ala Met Leu Val Gly Gln Val Thr
Leu Leu Asp Leu 1475 1480 1485Leu Lys Leu Thr Val Ala Val Gly Leu
His Phe His Glu Met Asn 1490 1495 1500Asn Gly Gly Asp Ala Met Tyr
Met Ala Leu Ile Ala Ala Phe Ser 1505 1510 1515Ile Arg Pro Gly Leu
Leu Ile Gly Phe Gly Leu Arg Thr Leu Trp 1520 1525 1530Ser Pro Arg
Glu Arg Leu Val Leu Thr Leu Gly Ala Ala Met Val 1535 1540 1545Glu
Ile Ala Leu Gly Gly Val Met Gly Gly Leu Trp Lys Tyr Leu 1550 1555
1560Asn Ala Val Ser Leu Cys Ile Leu Thr Ile Asn Ala Val Ala Ser
1565 1570 1575Arg Lys Ala Ser Asn Thr Ile Leu Pro Leu Met Ala Leu
Leu Thr 1580 1585 1590Pro Val Thr Met Ala Glu Val Arg Leu Ala Ala
Met Phe Phe Cys 1595 1600 1605Ala Met Val Ile Ile Gly Val Leu His
Gln Asn Phe Lys Asp Thr 1610 1615 1620Ser Met Gln Lys Thr Ile Pro
Leu Val Ala Leu Thr Leu Thr Ser 1625 1630 1635Tyr Leu Gly Leu Thr
Gln Pro Phe Leu Gly Leu Cys Ala Phe Leu 1640 1645 1650Ala Thr Arg
Ile Phe Gly Arg Arg Ser Ile Pro Val Asn Glu Ala 1655 1660 1665Leu
Ala Ala Ala Gly Leu Val Gly Val Leu Ala Gly Leu Ala Phe 1670 1675
1680Gln Glu Met Glu Asn Phe Leu Gly Pro Ile Ala Val Gly Gly Leu
1685 1690 1695Leu Met Met Leu Val Ser Val Ala Gly Arg Val Asp Gly
Leu Glu 1700 1705 1710Leu Lys Lys Leu Gly Glu Val Ser Trp Glu Glu
Glu Ala Glu Ile 1715 1720 1725Ser Gly Ser Ser Ala Arg Tyr Asp Val
Ala Leu Ser Glu Gln Gly 1730 1735 1740Glu Phe Lys Leu Leu Ser Glu
Glu Lys Val Pro Trp Asp Gln Val 1745 1750 1755Val Met Thr Ser Leu
Ala Leu Val Gly Ala Ala Leu His Pro Phe 1760 1765 1770Ala Leu Leu
Leu Val Leu Ala Gly Trp Leu Phe His Val Arg Gly 1775 1780 1785Ala
Arg Arg Ser Gly Asp Val Leu Trp Asp Ile Pro Thr Pro Lys 1790 1795
1800Ile Ile Glu Glu Cys Glu His Leu Glu Asp Gly Ile Tyr Gly Ile
1805 1810 1815Phe Gln Ser Thr Phe Leu Gly Ala Ser Gln Arg Gly Val
Gly Val 1820 1825 1830Ala Gln Gly Gly Val Phe His Thr Met Trp His
Val Thr Arg Gly 1835 1840 1845Ala Phe Leu Val Arg Asn Gly Lys Lys
Leu Ile Pro Ser Trp Ala 1850 1855 1860Ser Val Lys Glu Asp Leu Val
Ala Tyr Gly Gly Ser Trp Lys Leu 1865 1870 1875Glu Gly Arg Trp Asp
Gly Glu Glu Glu Val Gln Leu Ile Ala Ala 1880 1885
1890Val Pro Gly Lys Asn Val Val Asn Val Gln Thr Lys Pro Ser Leu
1895 1900 1905Phe Lys Val Arg Asn Gly Gly Glu Ile Gly Ala Val Ala
Leu Asp 1910 1915 1920Tyr Pro Ser Gly Thr Ser Gly Ser Pro Ile Val
Asn Arg Asn Gly 1925 1930 1935Glu Val Ile Gly Leu Tyr Gly Asn Gly
Ile Leu Val Gly Asp Asn 1940 1945 1950Ser Phe Val Ser Ala Ile Ser
Gln Thr Glu Val Lys Glu Glu Gly 1955 1960 1965Lys Glu Glu Leu Gln
Glu Ile Pro Thr Met Leu Lys Lys Gly Met 1970 1975 1980Thr Thr Val
Leu Asp Phe His Pro Gly Ala Gly Lys Thr Arg Arg 1985 1990 1995Phe
Leu Pro Gln Ile Leu Ala Glu Cys Ala Arg Arg Arg Leu Arg 2000 2005
2010Thr Leu Val Leu Ala Pro Thr Arg Val Val Leu Ser Glu Met Lys
2015 2020 2025Glu Ala Phe His Gly Leu Asp Val Lys Phe His Thr Gln
Ala Phe 2030 2035 2040Ser Ala His Gly Ser Gly Arg Glu Val Ile Asp
Ala Met Cys His 2045 2050 2055Ala Thr Leu Thr Tyr Arg Met Leu Glu
Pro Thr Arg Val Val Asn 2060 2065 2070Trp Glu Val Ile Ile Met Asp
Glu Ala His Phe Leu Asp Pro Ala 2075 2080 2085Ser Ile Ala Ala Arg
Gly Trp Ala Ala His Arg Ala Arg Ala Asn 2090 2095 2100Glu Ser Ala
Thr Ile Leu Met Thr Ala Thr Pro Pro Gly Thr Ser 2105 2110 2115Asp
Glu Phe Pro His Ser Asn Gly Glu Ile Glu Asp Val Gln Thr 2120 2125
2130Asp Ile Pro Ser Glu Pro Trp Asn Thr Gly His Asp Trp Ile Leu
2135 2140 2145Ala Asp Lys Arg Pro Thr Ala Trp Phe Leu Pro Ser Ile
Arg Ala 2150 2155 2160Ala Asn Val Met Ala Ala Ser Leu Arg Lys Ala
Gly Lys Ser Val 2165 2170 2175Val Val Leu Asn Arg Lys Thr Phe Glu
Arg Glu Tyr Pro Thr Ile 2180 2185 2190Lys Gln Lys Lys Pro Asp Phe
Ile Leu Ala Thr Asp Ile Ala Glu 2195 2200 2205Met Gly Ala Asn Leu
Cys Val Glu Arg Val Leu Asp Cys Arg Thr 2210 2215 2220Ala Phe Lys
Pro Val Leu Val Asp Glu Gly Arg Lys Val Ala Ile 2225 2230 2235Lys
Gly Pro Leu Arg Ile Ser Ala Ser Ser Ala Ala Gln Arg Arg 2240 2245
2250Gly Arg Ile Gly Arg Asn Pro Asn Arg Asp Gly Asp Ser Tyr Tyr
2255 2260 2265Tyr Ser Glu Pro Thr Ser Glu Asn Asn Ala His His Val
Cys Trp 2270 2275 2280Leu Glu Ala Ser Met Leu Leu Asp Asn Met Glu
Val Arg Gly Gly 2285 2290 2295Met Val Ala Pro Leu Tyr Gly Val Glu
Gly Thr Lys Thr Pro Val 2300 2305 2310Ser Pro Gly Glu Met Arg Leu
Arg Asp Asp Gln Arg Lys Val Phe 2315 2320 2325Arg Glu Leu Val Arg
Asn Cys Asp Leu Pro Val Trp Leu Ser Trp 2330 2335 2340Gln Val Ala
Lys Ala Gly Leu Lys Thr Asn Asp Arg Lys Trp Cys 2345 2350 2355Phe
Glu Gly Pro Glu Glu His Glu Ile Leu Asn Asp Ser Gly Glu 2360 2365
2370Thr Val Lys Cys Arg Ala Pro Gly Gly Ala Lys Lys Pro Leu Arg
2375 2380 2385Pro Arg Trp Cys Asp Glu Arg Val Ser Ser Asp Gln Ser
Ala Leu 2390 2395 2400Ser Glu Phe Ile Lys Phe Ala Glu Gly Arg Arg
Gly Ala Ala Glu 2405 2410 2415Val Leu Val Val Leu Ser Glu Leu Pro
Asp Phe Leu Ala Lys Lys 2420 2425 2430Gly Gly Glu Ala Met Asp Thr
Ile Ser Val Phe Leu His Ser Glu 2435 2440 2445Glu Gly Ser Arg Ala
Tyr Arg Asn Ala Leu Ser Met Met Pro Glu 2450 2455 2460Ala Met Thr
Ile Val Met Leu Phe Ile Leu Ala Gly Leu Leu Thr 2465 2470 2475Ser
Gly Met Val Ile Phe Phe Met Ser Pro Lys Gly Ile Ser Arg 2480 2485
2490Met Ser Met Ala Met Gly Thr Met Ala Gly Cys Gly Tyr Leu Met
2495 2500 2505Phe Leu Gly Gly Val Lys Pro Thr His Ile Ser Tyr Val
Met Leu 2510 2515 2520Ile Phe Phe Val Leu Met Val Val Val Ile Pro
Glu Pro Gly Gln 2525 2530 2535Gln Arg Ser Ile Gln Asp Asn Gln Val
Ala Tyr Leu Ile Ile Gly 2540 2545 2550Ile Leu Thr Leu Val Ser Ala
Val Ala Ala Asn Glu Leu Gly Met 2555 2560 2565Leu Glu Lys Thr Lys
Glu Asp Leu Phe Gly Lys Lys Asn Leu Ile 2570 2575 2580Pro Ser Ser
Ala Ser Pro Trp Ser Trp Pro Asp Leu Asp Leu Lys 2585 2590 2595Pro
Gly Ala Ala Trp Thr Val Tyr Val Gly Ile Val Thr Met Leu 2600 2605
2610Ser Pro Met Leu His His Trp Ile Lys Val Glu Tyr Gly Asn Leu
2615 2620 2625Ser Leu Ser Gly Ile Ala Gln Ser Ala Ser Val Leu Ser
Phe Met 2630 2635 2640Asp Lys Gly Ile Pro Phe Met Lys Met Asn Ile
Ser Val Ile Met 2645 2650 2655Leu Leu Val Ser Gly Trp Asn Ser Ile
Thr Val Met Pro Leu Leu 2660 2665 2670Cys Gly Ile Gly Cys Ala Met
Leu His Trp Ser Leu Ile Leu Pro 2675 2680 2685Gly Ile Lys Ala Gln
Gln Ser Lys Leu Ala Gln Arg Arg Val Phe 2690 2695 2700His Gly Val
Ala Lys Asn Pro Val Val Asp Gly Asn Pro Thr Val 2705 2710 2715Asp
Ile Glu Glu Ala Pro Glu Met Pro Ala Leu Tyr Glu Lys Lys 2720 2725
2730Leu Ala Leu Tyr Leu Leu Leu Ala Leu Ser Leu Ala Ser Val Ala
2735 2740 2745Met Cys Arg Thr Pro Phe Ser Leu Ala Glu Gly Ile Val
Leu Ala 2750 2755 2760Ser Ala Ala Leu Gly Pro Leu Ile Glu Gly Asn
Thr Ser Leu Leu 2765 2770 2775Trp Asn Gly Pro Met Ala Val Ser Met
Thr Gly Val Met Arg Gly 2780 2785 2790Asn His Tyr Ala Phe Val Gly
Val Met Tyr Asn Leu Trp Lys Met 2795 2800 2805Lys Thr Gly Arg Arg
Gly Ser Ala Asn Gly Lys Thr Leu Gly Glu 2810 2815 2820Val Trp Lys
Arg Glu Leu Asn Leu Leu Asp Lys Arg Gln Phe Glu 2825 2830 2835Leu
Tyr Lys Arg Thr Asp Ile Val Glu Val Asp Arg Asp Thr Ala 2840 2845
2850Arg Arg His Leu Ala Glu Gly Lys Val Asp Thr Gly Val Ala Val
2855 2860 2865Ser Arg Gly Thr Ala Lys Leu Arg Trp Phe His Glu Arg
Gly Tyr 2870 2875 2880Val Lys Leu Glu Gly Arg Val Ile Asp Leu Gly
Cys Gly Arg Gly 2885 2890 2895Gly Trp Cys Tyr Tyr Ala Ala Ala Gln
Lys Glu Val Ser Gly Val 2900 2905 2910Lys Gly Phe Thr Leu Gly Arg
Asp Gly His Glu Lys Pro Met Asn 2915 2920 2925Val Gln Ser Leu Gly
Trp Asn Ile Ile Thr Phe Lys Asp Lys Thr 2930 2935 2940Asp Ile His
Arg Leu Glu Pro Val Lys Cys Asp Thr Leu Leu Cys 2945 2950 2955Asp
Ile Gly Glu Ser Ser Ser Ser Ser Val Thr Glu Gly Glu Arg 2960 2965
2970Thr Val Arg Val Leu Asp Thr Val Glu Lys Trp Leu Ala Cys Gly
2975 2980 2985Val Asp Asn Phe Cys Val Lys Val Leu Ala Pro Tyr Met
Pro Asp 2990 2995 3000Val Leu Glu Lys Leu Glu Leu Leu Gln Arg Arg
Phe Gly Gly Thr 3005 3010 3015Val Ile Arg Asn Pro Leu Ser Arg Asn
Ser Thr His Glu Met Tyr 3020 3025 3030Tyr Val Ser Gly Ala Arg Ser
Asn Val Thr Phe Thr Val Asn Gln 3035 3040 3045Thr Ser Arg Leu Leu
Met Arg Arg Met Arg Arg Pro Thr Gly Lys 3050 3055 3060Val Thr Leu
Glu Ala Asp Val Ile Leu Pro Ile Gly Thr Arg Ser 3065 3070 3075Val
Glu Thr Asp Lys Gly Pro Leu Asp Lys Glu Ala Ile Glu Glu 3080 3085
3090Arg Val Glu Arg Ile Lys Ser Glu Tyr Met Thr Ser Trp Phe Tyr
3095 3100 3105Asp Asn Asp Asn Pro Tyr Arg Thr Trp His Tyr Cys Gly
Ser Tyr 3110 3115 3120Val Thr Lys Thr Ser Gly Ser Ala Ala Ser Met
Val Asn Gly Val 3125 3130 3135Ile Lys Ile Leu Thr Tyr Pro Trp Asp
Arg Ile Glu Glu Val Thr 3140 3145 3150Arg Met Ala Met Thr Asp Thr
Thr Pro Phe Gly Gln Gln Arg Val 3155 3160 3165Phe Lys Glu Lys Val
Asp Thr Arg Ala Lys Asp Pro Pro Ala Gly 3170 3175 3180Thr Arg Lys
Ile Met Lys Val Val Asn Arg Trp Leu Phe Arg His 3185 3190 3195Leu
Ala Arg Glu Lys Ser Pro Arg Leu Cys Thr Lys Glu Glu Phe 3200 3205
3210Ile Ala Lys Val Arg Ser His Ala Ala Ile Gly Ala Tyr Leu Glu
3215 3220 3225Glu Gln Glu Gln Trp Lys Thr Ala Asn Glu Ala Val Gln
Asp Pro 3230 3235 3240Lys Phe Trp Glu Leu Val Asp Glu Glu Arg Lys
Leu His Gln Gln 3245 3250 3255Gly Arg Cys Arg Thr Cys Val Tyr Asn
Met Met Gly Lys Arg Glu 3260 3265 3270Lys Lys Leu Ser Glu Phe Gly
Lys Ala Lys Gly Ser Arg Ala Ile 3275 3280 3285Trp Tyr Met Trp Leu
Gly Ala Arg Tyr Leu Glu Phe Glu Ala Leu 3290 3295 3300Gly Phe Leu
Asn Glu Asp His Trp Ala Ser Arg Glu Asn Ser Gly 3305 3310 3315Gly
Gly Val Glu Gly Ile Gly Leu Gln Tyr Leu Gly Tyr Val Ile 3320 3325
3330Arg Asp Leu Ala Ala Met Asp Gly Gly Gly Phe Tyr Ala Asp Asp
3335 3340 3345Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Ala Asp Leu
Asp Asp 3350 3355 3360Glu Gln Glu Ile Leu Asn Tyr Met Ser Pro His
His Lys Lys Leu 3365 3370 3375Ala Gln Ala Val Met Glu Met Thr Tyr
Lys Asn Lys Val Val Lys 3380 3385 3390Val Leu Arg Pro Ala Pro Gly
Gly Lys Ala Tyr Met Asp Val Ile 3395 3400 3405Ser Arg Arg Asp Gln
Arg Gly Ser Gly Gln Val Val Thr Tyr Ala 3410 3415 3420Leu Asn Thr
Ile Thr Asn Leu Lys Val Gln Leu Ile Arg Met Ala 3425 3430 3435Glu
Ala Glu Met Val Ile His His Gln His Val Gln Asp Cys Asp 3440 3445
3450Glu Ser Val Leu Thr Arg Leu Glu Ala Trp Leu Thr Glu His Gly
3455 3460 3465Cys Asn Arg Leu Lys Arg Met Ala Val Ser Gly Asp Asp
Cys Val 3470 3475 3480Val Arg Pro Ile Asp Asp Arg Phe Gly Leu Ala
Leu Ser His Leu 3485 3490 3495Asn Ala Met Ser Lys Val Arg Lys Asp
Ile Ser Glu Trp Gln Pro 3500 3505 3510Ser Lys Gly Trp Asn Asp Trp
Glu Asn Val Pro Phe Cys Ser His 3515 3520 3525His Phe His Glu Leu
Gln Leu Lys Asp Gly Arg Arg Ile Val Val 3530 3535 3540Pro Cys Arg
Glu Gln Asp Glu Leu Ile Gly Arg Gly Arg Val Ser 3545 3550 3555Pro
Gly Asn Gly Trp Met Ile Lys Glu Thr Ala Cys Leu Ser Lys 3560 3565
3570Ala Tyr Ala Asn Met Trp Ser Leu Met Tyr Phe His Lys Arg Asp
3575 3580 3585Met Arg Leu Leu Ser Leu Ala Val Ser Ser Ala Val Pro
Thr Ser 3590 3595 3600Trp Val Pro Gln Gly Arg Thr Thr Trp Ser Ile
His Gly Lys Gly 3605 3610 3615Glu Trp Met Thr Thr Glu Asp Met Leu
Glu Val Trp Asn Arg Val 3620 3625 3630Trp Ile Thr Asn Asn Pro His
Met Gln Asp Lys Thr Met Val Lys 3635 3640 3645Lys Trp Arg Asp Val
Pro Tyr Leu Thr Lys Arg Gln Asp Lys Leu 3650 3655 3660Cys Gly Ser
Leu Ile Gly Met Thr Asn Arg Ala Thr Trp Ala Ser 3665 3670 3675His
Ile His Leu Val Ile His Arg Ile Arg Thr Leu Ile Gly Gln 3680 3685
3690Glu Lys Tyr Thr Asp Tyr Leu Thr Val Met Asp Arg Tyr Ser Val
3695 3700 3705Asp Ala Asp Leu Gln Leu Gly Glu Leu Ile 3710
37159508PRTHuman immunodeficiency virus 9Met Gly Ala Arg Ala Ser
Val Leu Ser Gly Gly Lys Leu Asp Lys Trp1 5 10 15Glu Lys Ile Arg Leu
Arg Pro Gly Gly Lys Lys Thr Tyr Gln Leu Lys 20 25 30His Ile Val Trp
Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45Gly Leu Leu
Glu Thr Gly Gly Gly Cys Lys Gln Ile Leu Val Gln Leu 50 55 60Gln Pro
Ser Leu Gln Thr Gly Ser Glu Glu Leu Lys Ser Leu Tyr Asn65 70 75
80Ala Val Ala Thr Leu Tyr Cys Val His Gln Gly Ile Glu Val Arg Asp
85 90 95Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Glu Gln Asn Lys Ser
Lys 100 105 110Lys Lys Ala Gln Gln Ala Ala Ala Asp Thr Gly Asn Ser
Ser Gln Val 115 120 125Ser Gln Asn Tyr Pro Ile Val Gln Asn Leu Gln
Gly Gln Met Val His 130 135 140Gln Ala Ile Ser Pro Arg Thr Leu Asn
Ala Trp Val Lys Val Ile Glu145 150 155 160Glu Lys Ala Phe Ser Pro
Glu Val Ile Pro Met Phe Ser Ala Leu Ser 165 170 175Glu Gly Ala Thr
Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly 180 185 190Gly His
Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu 195 200
205Ala Ala Glu Trp Asp Arg Leu His Pro Ala His Ala Gly Pro Asn Ala
210 215 220Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly
Thr Thr225 230 235 240Ser Thr Leu Gln Glu Gln Ile Gly Trp Met Thr
Ser Asn Pro Pro Val 245 250 255Pro Val Gly Glu Ile Tyr Lys Arg Trp
Ile Ile Leu Gly Leu Asn Lys 260 265 270Ile Val Arg Met Tyr Ser Pro
Val Ser Ile Leu Asp Ile Arg Gln Gly 275 280 285Pro Lys Glu Pro Phe
Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu 290 295 300Arg Ala Glu
Gln Ala Ser Gln Asp Val Lys Asn Trp Met Thr Glu Thr305 310 315
320Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala
325 330 335Leu Gly Pro Ala Ala Thr Leu Glu Glu Met Met Thr Ala Cys
Gln Gly 340 345 350Val Gly Gly Pro Ser His Lys Ala Arg Ile Leu Ala
Glu Ala Met Ser 355 360 365Gln Val Thr Ser Pro Ala Asn Ile Met Met
Gln Arg Gly Asn Phe Arg 370 375 380Asn Gln Arg Lys Thr Ile Lys Cys
Phe Asn Cys Gly Lys Glu Gly His385 390 395 400Leu Ala Arg His Cys
Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys 405 410 415Gly Arg Glu
Gly His Gln Met Lys Asp Cys Thr Glu Arg Gln Ala Asn 420 425 430Phe
Leu Gly Lys Ile Trp Pro Ser His Lys Gly Arg Pro Gly Asn Phe 435 440
445Leu Gln Ser Arg Pro Glu Pro Thr Ala Pro Pro Glu Glu Ser Phe Arg
450 455 460Phe Gly Glu Glu Thr Thr Thr Pro Pro Gln Lys Gln Glu Pro
Leu Pro465 470 475 480Ser Gln Lys Gln Glu Thr Ile Asp Lys Asp Leu
Tyr Pro Leu Ala Ser 485 490 495Leu Lys Ser Leu Phe Gly Asn Asp Pro
Ser Leu Gln 500 505
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